U.S. patent application number 10/321188 was filed with the patent office on 2003-09-25 for compositions and methods for hydroxylating epothilones.
Invention is credited to Basch, Jonathan David, Chiang, Shu-Jen David, Liu, Suo-Win, Nayeem, Akbar, Sun, Yuhua, You, Li.
Application Number | 20030180760 10/321188 |
Document ID | / |
Family ID | 32710750 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180760 |
Kind Code |
A1 |
Basch, Jonathan David ; et
al. |
September 25, 2003 |
Compositions and methods for hydroxylating epothilones
Abstract
Isolated nucleic acid sequences and polypeptides encoded thereby
for epothilone B hydroxylase and mutants and variants thereof and a
ferredoxin located downstream from the epothilone B hydroxylase
gene are provided. Also provided are vectors and cells containing
these vectors. In addition, methods for producing recombinant
microorganisms, methods for using these recombinant microorganism
to produce hydroxyalkyl-bearing epothilones and an epothilone
analog produced by a mutant of epothilone B hydroxylase are
provided.
Inventors: |
Basch, Jonathan David;
(DeWitt, NY) ; Chiang, Shu-Jen David; (Manlius,
NY) ; Liu, Suo-Win; (Manlius, NY) ; Nayeem,
Akbar; (Newtown, PA) ; Sun, Yuhua; (East
Syracuse, NY) ; You, Li; (Jamesville, NY) |
Correspondence
Address: |
STEPHEN B. DAVIS
BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Family ID: |
32710750 |
Appl. No.: |
10/321188 |
Filed: |
December 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60344271 |
Dec 26, 2001 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/118; 435/191; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07K 14/79 20130101;
C07K 2299/00 20130101; C12P 17/181 20130101; C12N 9/0077
20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/191; 435/320.1; 435/252.3; 435/118; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 017/16; C12N 009/06; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid sequence encoding epothilone B
hydrolase or a mutant or variant thereof.
2. The isolated nucleic acid sequence of claim 1 comprising SEQ ID
NO: 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68,
72 or 74.
3. The isolated nucleic acid sequence of claim 1 comprising SEQ ID
NO:1.
4. The isolated nucleic acid sequence of claim 1 encoding a mutant
with at least one amino acid substitution in an active site of the
epothilone B hydroxylase enzyme.
5. The isolated nucleic acid sequence of claim 1 encoding a mutant
with at least one amino acid substitution at amino acid GLU31,
ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190,
GLU 231, SER294, PHE237, or ILE365 of SEQ ID NO:2.
6. The isolated nucleic acid sequence of claim 1 encoding a mutant
with at least one amino acid substitution at amino acid LEU39,
GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66,
ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79,
ILE80, ASP84, LYS85, PRO86, PHE87, ARG88, PRO89, SER90, LEU91,
ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169,
GLN170, CYS172, SER173, SER174, ARG175, MET176, LEU177, SER178,
ARG179, ARG186, PHE190, LEU193, VAL233, GLY234, LEU235, ALA236,
PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244,
GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, THR287,
ILE288, ALA289, GLU290, THR291, ALA292, THR293, SER294, ARG295,
PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316,
VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352,
CYS353, LEU354, GLY355, GLN356, LEU358, ALA359, GLU362, LYS389,
ASP391, SER392, THR393, ILE394, or TYR395 of SEQ ID NO:2.
7. The isolated nucleic acid sequence of claim 1 encoding a variant
comprising SEQ ID NO:43, 44, 45, 46, 47, 48 or 49.
8. A polypeptide encoded by the isolated nucleic acid sequence of
claim 1.
9. An isolated nucleic acid molecule that is capable of hybridizing
to a nucleic acid sequence of claim 2, or to the complementary
sequence of said nucleic acid sequence, under hybridization
conditions of 3.times.SSC at 65.degree. C. for 16 hours, said
isolated nucleic acid molecule being capable of remaining
hybridized to said nucleic acid sequence, or to the complementary
sequence of said nucleic acid sequence, under wash conditions of
0.5.times.SSC, 55.degree. C. for 30 minutes.
10. An isolated polypeptide comprising SEQ ID NO:2.
11. An isolated mutant polypeptide of epothilone B hydroxylase of
SEQ ID NO:2 comprising an amino acid sequence with at least one
amino acid substitution in an active site of epothilone B
hydroxylase enzyme of SEQ ID NO:2.
12. An isolated mutant polypeptide of epothilone B hydroxylase of
SEQ ID NO:2 comprising an amino acid sequence with at least one
amino acid substitution at amino acid GLU31, ARG67, ARG88, ILE92,
ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU 231, SER294,
PHE237, or ILE365 of SEQ ID NO:2.
13. An isolated mutant polypeptide of epothilone B hydroxylase of
SEQ ID NO:2 comprising an amino acid sequence with at least one
amino acid substitution at amino acid LEU39, GLN43, ALA45, MET57,
LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69,
LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, LYS85,
PRO86, PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94,
ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172,
SER173, SER174, ARG175, MET176, LEU177, SER178, ARG179, ARG186,
PHE190, LEU193, VAL233, GLY234, LEU235, ALA236, PHE237, LEU238,
LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246,
THR247, ALA248, ASN249, MET250, LEU283, THR287, ILE288, ALA289,
GLU290, THR291, ALA292, THR293, SER294, ARG295, PHE296, ALA297,
THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345,
PHE346, GLY347, PHE348, VAL350, HIS351, GLN352, CYS353, LEU354,
GLY355, GLN356, LEU358, ALA359, GLU362, LYS389, ASP391, SER392,
THR393, ILE394, or TYR395 of SEQ ID NO:2.
14. An isolated mutant polypeptide of epothilone B hydroxylase
comprising SEQ ID NO: 31, 33, 35, 61, 63, 65, 67, 69, 71, 73 or
75.
15. An isolated variant polypeptide of epothilone B hydroxylase
comprising SEQ ID NO: 43, 44, 45, 46, 47, 48 or 49.
16. An isolated nucleic acid sequence encoding a ferredoxin.
17. The isolated nucleic acid sequence of claim 16 comprising SEQ
ID NO:3.
18. A polypeptide encoded by the isolated nucleic acid sequence of
claim 16.
19. An isolated nucleic acid molecule that is capable of
hybridizing to the nucleic acid sequence set forth in SEQ ID NO:3,
or to the complementary sequence of the nucleic acid sequence set
forth in SEQ ID NO:3, under hybridization conditions of 3.times.SSC
at 65.degree. C. for 16 hours, said isolated nucleic acid molecule
being capable of remaining hybridized to the nucleic acid sequence
set forth in SEQ ID NO:3, or to the complementary sequence of the
nucleic acid sequence set forth in SEQ ID NO:3, under wash
conditions of 0.5.times.SSC, 55.degree. C. for 30 minutes.
20. A vector comprising the isolated nucleic acid sequence of claim
1.
21. The vector of claim 20 further comprising an isolated nucleic
acid sequence encoding a ferredoxin.
22. A host cell comprising the vector of claim 20.
23. A host cell comprising the vector of claim 21.
24. A method for producing recombinant microorganisms which
hydroxylate epothilones having a terminal alkyl group to produce
epothilones having a terminal hydroxyalkyl group, said method
comprising transfecting a microorganism with the vector of claim 20
or 21.
25. A recombinantly produced microorganism that hydroxylates
epothilones having a terminal alkyl group to produce epothilones
having a terminal hydroxyalkyl group.
26. The recombinantly produced microorganism of claim 25 wherein
said microorganism expresses a nucleic acid sequence of SEQ ID NO:
1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 72
or 74.
27. A method for the preparation of at least one epothilone of the
following formula
IHO--CH.sub.2--(A.sub.1).sub.n--(Q).sub.m--(A.sub.2).su- b.o--E
(I)where A.sub.1 and A.sub.2 are independently selected from the
group of optionally substituted C.sub.1-C.sub.3 alkyl and alkenyl;
Q is an optionally substituted ring system containing one to three
rings and at least one carbon to carbon double bond in at least one
ring; n, m, and o are integers selected from the group consisting
of zero and 1, where at least one of m or n or o is 1; and E is an
epothilone core; comprising the steps of contacting at least one
epothilone of the following formula
IICH.sub.3--(A.sub.1).sub.n--(Q).sub.m--(A.sub.2).sub.o--E
(II)where A.sub.1, Q, A.sub.2, E, n, m, and o are defined as above;
with a recombinantly produced microorganism, or an enzyme derived
therefrom, which is capable of selectively catalyzing the
hydroxylation of Formula II, and effecting said hydroxylation.
28. A method for the preparation of an epothilone analog of Formula
A 6said method comprising biotransforming epothilone B to the
epothilone analog of Formula A by incubation with a mutant
epothilone B hydroxylase enzyme comprising SEQ ID NO:31.
29. A compound of Formula A 7or a pharmaceutically acceptable salt
thereof.
30. A homology model of epothilone B hydroxylase having a root mean
square deviation of conserved residue backbone atoms of less than
about 4.0 .ANG. when superimposed on a corresponding backbone atoms
described by structure coordinates listed in Appendix 1.
31. A method for producing a mutant with altered biological
properties, function, yield of a desired product, rate of reaction,
substrate specificity, or activity as compared to epothilone B
hydroxylase, said method comprising the steps of: identifying an
amino acid of SEQ ID NO:2 to mutate; and mutating the identified
amino acid to create a mutant protein.
32. The method of claim 31 wherein a homology model of epothilone B
hydroxylase having a root mean square deviation of conserved
residue backbone atoms of less than about 4.0 .ANG. when
superimposed on a corresponding backbone atoms described by
structure coordinates listed in Appendix 1 is used to identify an
amino acid of SEQ ID NO: 2 to mutate.
33. The method of claim 31 wherein the identified amino acid is
LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65,
ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78,
GLN79, ILE80, ASP84, LYS85, PRO86, PHE87, ARG88, PRO89, SER90,
LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155,
PHE169, GLN170, CYS172, SER173, SER174, ARG175, MET176, LEU177,
SER178, ARG179, ARG186, PHE190, LEU193, VAL233, GLY234, LEU235,
ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243,
HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283,
THR287, ILE288, ALA289, GLU290, THR291, ALA292, THR293, SER294,
ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315,
GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351,
GLN352, CYS353, LEU354, GLY355, GLN356, LEU358, ALA359, GLU362,
LYS389, ASP391, SER392, THR393, ILE394, or TYR395 of SEQ ID
NO:2.
34. The method of claim 31 wherein the identified amino acid is
GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176,
PHE190, GLU 231, SER294, PHE237, or ILE365 of SEQ ID NO:2.
35. The method of claim 31 wherein the mutant protein improves
yield of a desired product as compared to the yield of a desired
product obtained using epothilone B hydroxylase.
36. The method of claim 35 wherein the desired product is
epothilone F.
37. The method of claim 31 wherein the mutant improves the rate of
reaction as compared to the rate of reaction using epothilone B
hydroxylase.
38. The method of claim 31 wherein the mutant exhibits altered
substrate specificity as compared to substrate specificity of
epothilone B hydroxylase.
39. The method of claim 38 wherein amino acid SER294 is
mutated.
40. The method of claim 31 wherein the mutant exhibits essentially
similar biological activity or function to epothilone B
hydroxylase.
41. A machine-readable data storage medium comprising a data
storage material encoded with structure coordinates set forth in
Appendix 1.
Description
BASIS FOR PRIORITY CLAIM
[0001] This application claims the benefit of priority from U.S.
Provisional Application No. 60/344,271, filed Dec. 26, 2001, which
is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to isolated nucleic acids
sequences and polypeptides encoded thereby for epothilone B
hydroxylase and mutants and variants thereof, and a ferredoxin
located downstream from the epothilone B hydroxylase gene. The
present invention also relates to recombinant microorganisms
expressing epothilone B hydroxylase or a mutant or variant thereof
and/or ferredoxin which are capable of hydroxylating small organic
molecule compounds, such as epothilones, having a terminal alkyl
group to produce compounds having a terminal hydroxyalkyl group.
Also provided are methods for recombinantly producing such
microorganisms as well as methods for using these recombinant
microorganisms in the synthesis of compounds having a terminal
hydroxylalkyl group. The compositions and methods of the present
invention are useful in preparation of epothilones having a variety
of utilities in the pharmaceutical field. A novel epothilone analog
produced using a mutant of epothilone B hydroxylase of the present
invention is also described.
BACKGROUND OF THE INVENTION
[0003] Epothilones are macrolide compounds that find utility in the
pharmaceutical field. For example, epothilones A and B having the
structures: 1
1 Epothilone A R = H Epothilone B R = Me
[0004] have been found to exert microtubule-stabilizing effects
similar to paclitaxel (TAXOL.RTM.) and hence cytotoxic activity
against rapidly proliferating cells, such as, tumor cells or cells
associated with other hyperproliferative cellular diseases, see
Bollag et al., Cancer Res., Vol. 55, No. 11, 2325-2333 (1995).
[0005] Epothilones A and B are natural anticancer agents produced
by Sorangium cellulosum that were first isolated and characterized
by Hofle et al., DE 4138042; WO 93/10121; Angew. Chem. Int. Ed.
Engl. Vol. 35, No13/14, 1567-1569 (1996); and J. Antibiot., Vol.
49, No. 6, 560-563 (1996). Subsequently, the total syntheses of
epothilones A and B have been published by Balog et al., Angew.
Chem. Int. Ed. Engl., Vol. 35, No. 23/24, 2801-2803, 1996; Meng et
al., J. Am. Chem. Soc., Vol. 119, No. 42, 10073-10092 (1997);
Nicolaou et al., J. Am. Chem. Soc., Vol. 119, No. 34, 7974-7991
(1997); Schinzer et al., Angew. Chem. Int. Ed. Eng., Vol. 36, No.
5, 523-524 (1997); and Yang et al., Angew. Chem. Int. Ed. Engl.,
Vol. 36, No. 1/2, 166-168, 1997. WO 98/25929 disclosed the methods
for chemical synthesis of epothilone A, epothilone B, analogs of
epothilone and libraries of epothilone analogs. The structure and
production from Sorangium cellulosum DSM 6773 of epothilones C, D,
E, and F was disclosed in WO 98/22461. FIG. 1 provides a diagram of
the biotransformation as described in WO 00/39276 of epothilone B
to epothilone F in Actinomycetes species strain SC15847 (ATCC
PT-1043), subsequently identified as Amycolatopsis orientalis.
[0006] Cytochrome P450 enzymes are found in prokaryotes and
eukaryotic cells and have in common a heme binding domain which can
be distinguished by an absorbance peak at 450 nm when complexed
with carbon monoxide. Cytochrome P450 enzymes perform a broad
spectrum of oxidative reactions on primarily hydrophobic substrates
including aromatic and benzylic rings, and alkanes. In prokaryotes
they are found as detoxifying systems and as a first enzymatic step
in metabolizing substrates such as toluene, benzene and camphor.
Cytochrome P450 genes have also been found in biosynthetic pathways
of secondary metabolites such as nikkomycin in Streptomyces tendae
(Bruntner, C. et al, 1999, Mol. Gen. Genet. 262: 102-114),
doxorubicin (Dickens, M. L, Strohl, W. R., 1996, J. Bacteriol, 178:
3389-3395) and in the epothilone biosynthetic cluster of Sorangium
cellulosum (Julien, B. et al., 2000, Gene, 249: 153-160). With a
few exceptions, the cytochrome P450 systems in prokaryotes are
composed of three proteins; a ferredoxin NADH or NADPH dependent
reductase, an iron-sulfur ferredoxin and the cytochrome P450 enzyme
(Lewis, D. F., Hlavica, P., 2000, Biochim. Biophys. Acta., 1460:
353-374). Electrons are transferred from ferredoxin reductase to
the ferredoxin and finally to the cytochrome P450 enzyme for the
splitting of molecular oxygen.
SUMMARY OF THE INVENTION
[0007] An object of the present invention is to provide isolated
nucleic acid sequences encoding epothilone B hydroxylase and
variants or mutants thereof and isolated nucleic acid sequences
encoding ferredoxin or variants or mutants thereof.
[0008] Another object of the present invention is to provide
isolated polypeptides comprising amino acid sequences of epothilone
B hydroxylase and variants or mutants thereof and isolated
polypeptides comprising amino acid sequences of ferredoxin and
variants or mutants thereof.
[0009] Another object of the present invention is to provide
structure coordinates of the homology model of the epothilone B
hydroxylase. The structure coordinates are listed in Appendix 1.
This model of the present invention provides a means for designing
modulators of a biological function of epothilone B hydroxylase as
well as additional mutants of epothilone B hydroxylase with altered
specificities.
[0010] Another object of the present invention is to provide
vectors comprising nucleic acid sequences encoding epothilone B
hydroxylase or a variant or mutant thereof and/or ferredoxin or a
variant or mutant thereof. In a preferred embodiment, these vectors
further comprise a nucleic acid sequence encoding ferredoxin.
[0011] Another object of the present invention is to provide host
cells comprising a vector containing a nucleic acid sequence
encoding epothilone B hydroxylase or a variant or mutant thereof
and/or ferredoxin or a variant or mutant thereof.
[0012] Another object of the present invention is to provide a
method for producing recombinant microorganisms that are capable of
hydroxylating compounds, and in particular epothilones, having a
terminal alkyl group to produce compounds having a terminal
hydroxyalkyl group.
[0013] Another object of the present invention is to provide
microorganisms produced recombinantly which are capable of
hydroxylating compounds, and in particular epothilones, having a
terminal alkyl group to produce compounds having a terminal
hydroxyalkyl group.
[0014] Another object of the present invention is to provide
methods for hydroxylating compounds in these recombinant
microorganisms. In particular, the present invention provides a
method for the preparation of hydroxyalkyl-bearing epothilones,
which compounds find utility as antitumor agents and as starting
materials in the preparation of other epothilone analogs.
[0015] Yet another object of the present invention is to provide a
compound of Formula A: 2
[0016] referred to herein as 24-OH epothilone B or 24-OH EpoB, as
well as compositions and methods for production of compositions
comprising the compound of Formula A.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 provides a schematic of the biotransformation as set
forth in WO 00/39276, U.S. application Ser. No. 09/468,854, filed
Dec. 21, 1999, of epothilone B to epothilone F by Amycolatopsis
orientalis strain SC15847 (PTA1043).
[0018] FIG. 2 shows the nucleic acid sequence alignments of SEQ ID
NO:5 through SEQ ID NO:22 used to design the PCR primers for
cloning of the nucleic acid sequence encoding epothilone B
hydroxylase.
[0019] FIG. 3 shows the sequence alignment between epothilone B
hydroxylase (SEQ ID NO:2) and EryF (PDB code 1JIN chain A; SEQ ID
NO:76). The asterisks indicate sequence identities, the colons (:)
similar residues.
[0020] FIG. 4 provides a homology model of epothilone B hydroxylase
based upon sequence alignment with EryF as shown in FIG. 3.
[0021] FIG. 5 shows an energy plot of the epothilone B hydroxylase
model (indicated by dashed line) relative to EryF (PDB code 1JIN;
indicated by solid line). An averaging window size of 51 residues
was used, i.e., the energy at a given residue position is
calculated as the average of the energies of the 51 residues in the
sequence that lie with the given residue at the central
positions.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to isolated nucleic acid
sequences and polypeptides and methods for obtaining compounds with
desired substituents at a terminal carbon position. In particular,
the present invention provides compositions and methods for the
preparation of hydroxyalkyl-bearing epothilones, which compounds
find utility as antitumor agents and as starting materials in the
preparation of other epothilone analogs.
[0023] The term "epothilone," as used herein, denotes compounds
containing an epothilone core and a side chain group as defined
herein. The term "epothilone core," as used herein, denotes a
moiety containing the core structure (with the numbering of ring
system positions used herein shown): 3
[0024] wherein the substituents are as follows:
[0025] Q is selected from the group consisting of 4
[0026] W is O or NR.sub.6;
[0027] X is selected from the group consisting of O, H and
OR.sub.7;
[0028] M is O, S, NR.sub.8, CR.sub.9R.sub.10;
[0029] B.sub.1 and B.sub.2 are selected from the group consisting
of OR.sub.11, OCOR.sub.12;
[0030] R.sub.1-R.sub.5 and R.sub.12-R.sub.17 are selected from the
group consisting of H, alkyl, substituted alkyl, aryl, and
heterocyclo, and wherein R.sub.1 and R.sub.2 are alkyl they can be
joined to form a cycloalkyl;
[0031] R.sub.6 is selected from the group consisting of H, alkyl,
and substituted alkyl;
[0032] R.sub.7 and R.sub.11 are selected from the group consisting
of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl and
dialkylarylsilyl;
[0033] R.sub.8 is selected from the group consisting of H, alkyl,
substituted alkyl, R.sub.13C.dbd.O, R.sub.14OC.dbd.O and
R.sub.15SO.sub.2; and
[0034] R.sub.9 and R.sub.10 are selected from the group consisting
of H, halogen, alkyl, substituted alkyl, aryl, heterocyclo,
hydroxy, R.sub.16C.dbd.O, and R.sub.17OC.dbd.O.
[0035] The term "side chain group" refers to substituent G as
defined above for Epothilone A or B or G.sub.1 and G.sub.2 as shown
below.
[0036] G.sub.1 is the following formula V
HO--CH.sub.2--(A.sub.1).sub.n--(Q).sub.m--(A.sub.2).sub.o (V),
[0037] and
[0038] G.sub.2 is the following formula VI
CH.sub.3--(A.sub.1).sub.n--(Q).sub.m--(A.sub.2).sub.o (VI),
[0039] where
[0040] A.sub.1 and A.sub.2 are independently selected from the
group of optionally substituted C.sub.1-C.sub.3 alkyl and
alkenyl;
[0041] Q is optionally substituted ring system containing one to
three rings and at least one carbon to carbon double bond in at
least one ring; and
[0042] n, m, and o are integers independently selected from the
group consisting of zero and 1, where at least one of m, n or o is
1.
[0043] The term "terminal carbon" or "terminal alkyl group" refers
to the terminal carbon or terminal methyl group of the moiety
either directly bonded to the epothilone core at position 15 or to
the terminal carbon or terminal alkyl group of the side chain group
bonded at position 15. It is understood that the term "alkyl group"
includes alkyl and substituted alkyl as defined herein.
[0044] The term "alkyl" refers to optionally substituted, straight
or branched chain saturated hydrocarbon groups of 1 to 20 carbon
atoms, preferably 1 to 7 carbon atoms. The expression "lower alkyl"
refers to optionally substituted alkyl groups of 1 to 4 carbon
atoms.
[0045] The term "substituted alkyl" refers to an alkyl group
substituted by, for example, one to four substituents, such as,
halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy,
cycloalkyloxy, heterocyclooxy, oxo, alkanoyl, aryloxy, alkanoyloxy,
amino, alkylamino, arylamino, aralkylamino, cycloalkylamino,
heterocycloamino, disubstituted amines in which the 2 amino
substituents are selected from alkyl, aryl or aralkyl,
alkanoylamino, aroylamino, aralkanoylamino, substituted
alkanoylamino, substituted arylamino, substituted aralkanoylamino,
thiol, alkylthio, arylthio, aralkylthio, cycloalkylthio,
heterocyclothio, alkylthiono, arylthiono, aralkylthiono,
alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, sulfonamido (e.g.
SO.sub.2NH.sub.2), substituted sulfonamido, nitro, cyano, carboxy,
carbamyl (e.g. CONH.sub.2), substituted carbamyl (e.g. CONH alkyl,
CONH aryl, CONH aralkyl or cases where there are two substituents
on the nitrogen selected from alkyl, aryl or aralkyl),
alkoxycarbonyl, aryl, substituted aryl, guanidino and heterocyclos,
such as, indolyl, imidazolyl, furyl, thienyl, thiazolyl,
pyrrolidyl, pyridyl, pyrimidyl and the like. Where noted above
where the substituent is further substituted it will be with
halogen, alkyl, alkoxy, aryl or aralkyl.
[0046] In accordance with one aspect of the present invention there
are provided isolated polynucleotides that encode epothilone B
hydroxylase, an enzyme capable of hydroxylating epothilones having
a terminal alkyl group to produce epothilones having a terminal
hydroxyalkyl group.
[0047] In accordance with another aspect of the present invention
there are provided isolated polynucleotides that encode a
ferredoxin, the gene for which is located downstream from the
epothilone B hydroxylase gene. Ferredoxin is a protein of the
cytochrome P450 system.
[0048] By "polynucleotides", as used herein, it is meant to include
any form of DNA or RNA such as cDNA or genomic DNA or mRNA,
respectively, encoding these enzymes or an active fragment thereof
which are obtained by cloning or produced synthetically by well
known chemical techniques. DNA may be double- or single-stranded.
Single-stranded DNA may comprise the coding or sense strand or the
non-coding or antisense strand. Thus, the term polynucleotide also
includes polynucleotides exhibiting at least 60% or more,
preferably at least 80%, homology to sequences disclosed herein,
and which hybridize under stringent conditions to the
above-described polynucleotides. As used herein, the term
"stringent conditions" means hybridization conditions of 60.degree.
C. at 2.times.SSC buffer. More preferred are isolated nucleic acid
molecules capable of hybridizing to the nucleic acid sequence set
forth in 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66,
68, 70, 72, or 74 or SEQ ID NO:3, or to the complementary sequence
of the nucleic acid sequence set forth in SEQ ID NO:1, 30, 32, 34,
36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72 ,or 74 or
SEQ ID NO:3, under hybridization conditions of 3.times.SSC at
65.degree. C. for 16 hours, and which are capable of remaining
hybridized to the nucleic acid sequence set forth in SEQ ID NO:1,
30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72
or 74 or SEQ ID NO:3, or to the complementary sequence of the
nucleic acid sequence set forth in SEQ ID NO:1, 30, 32, 34, 36, 37,
38, 39, 40, 41 or 42, 60, 62, 64, 66, 68, 70, 72 or 74 or SEQ ID
NO:3, under wash conditions of 0.5.times.SSC, 55.degree. C. for 30
minutes.
[0049] In one embodiment, a polynucleotide of the present invention
comprises the genomic DNA depicted in SEQ ID NO:1 or a homologous
sequence or fragment thereof which encodes a polypeptide having
similar activity to that of this epothilone B hydroxylase.
Alternatively, a polynucleotide of the present invention may
comprise the genomic DNA depicted in SEQ ID NO:3 or a homologous
sequence or fragment thereof which encodes a polypeptide having
similar activity to this ferredoxin. Due to the degeneracy of the
genetic code, polynucleotides of the present invention may also
comprise other nucleic acid sequences encoding this enzyme and
derivatives, variants or active fragments thereof.
[0050] The present invention also relates to variants of these
polynucleotides which may be naturally occurring, i.e., present in
microorganisms such as Amycolatopsis orientalis and Amycolata
autotrophica, or in soil or other sources from which nucleic acids
can be isolated, or mutants prepared by well known mutagenesis
techniques. Exemplary variants polynucleotides of the present
invention are depicted in SEQ ID NO: 36-42.
[0051] By "mutants" as used herein it is meant to be inclusive of
nucleic acid sequences with one or more point mutations, or
deletions or additions of nucleic acids as compared to SEQ ID NO: 1
or 3, but which still encode a polypeptide or fragment with similar
activity to the polypeptides encoded by SEQ ID NO: 1 or 3. In a
preferred embodiment, mutations are made which alter the substrate
specificity and/or yield of the enzyme. A preferred region of
mutation with respect to the epothilone B hydroxylase gene is that
region of the nucleic acid sequence coding for the approximately
113 amino acids residues comprising the active site of the enzyme.
Also preferred are mutants encoding a polypeptide with at least one
amino acid substitution at amino acid position GLU31, ARG67, ARG88,
ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU 231,
SER294, PHE237, or ILE365 of SEQ ID NO:1. Exemplary polynucleotide
mutants of the present invention are depicted in SEQ ID NO: 30, 32,
34, 60, 62, 64, 66, 68, 70, 72 and 74.
[0052] Cloning of the nucleic acid sequence of SEQ ID NO:1 encoding
epothilone B hydroxylase was performed using PCR primers designed
by aligning the nucleic acid sequences of six cytochrome P450 genes
from bacteria. The following cytochrome P450 genes were
aligned:
[0053] Sequence 1: Locus: STMSUACB; Accession number: M32238;
Reference: Omer, C. A., J. Bacteriol. 172: 3335-3345 (1990)
[0054] Sequence 2: Locus: STMSUBCB; Accession number: M32239;
Reference: Omer, C. A., J. Bacteriol. 172: 3335-3345 (1990)
[0055] Sequence 3: Locus: AB018074 (formerly STMORFA); Accession
number: AB018074; Reference: Ueda, K., J. Antibiot. 48: 638-646
(1995)
[0056] Sequence 4: Locus: SSU65940; Accession number: U65940;
Reference: Motamedi, H., J. Bacteriol. 178: 5243-5248 (1996)
[0057] Sequence 5: Locus: STMOLEP; Accession number: L37200;
Reference: Rodriguez, A. M., FEMS Microbiol. Lett. 127: 117-120
(1995)
[0058] Sequence 6: Locus: SERCP450A; Accession number: M83110;
Reference: Andersen, J. F. and Hutchinson, C. R., J. Bacteriol.
174: 725-735 (1992)
[0059] Alignments were performed using an implementation of the
algorithm of Myers, E. W. and W. Miller. 1988. CABIOS 4:1, 11-17.,
the Align program from Scientific and Educational Software (Durham,
N.C., USA). Three highly conserved regions were identified in the
I-helix, containing the oxygen binding domain, in the K-helix, and
spanning the B-bulge and L-helix containing the conserved heme
binding domain. Primers were designed to the three conserved
regions identified in the alignment. Primers P450-1.sup.+ (SEQ ID
NO:23) and P450-1a.sup.+ (SEQ ID NO:24) were designed from the I
helix, Primer P450-2.sup.+ (SEQ ID NO:25) was designed from the
B-Bulge and L-helix region and Primer P450-3.sup.- (SEQ ID NO:27)
was designed as the reverse complement to the heme binding
protein.
[0060] Genomic fragments were then amplified via polymerase chain
reaction (PCR). After PCR amplification, the reaction products were
separated by gel electrophoresis and fragments of the expected size
were excised. The DNA was extracted from the agarose gel slices
using the Qiaquick gel extraction procedure (Qiagen, Santa Clarita,
Calif., USA). The fragments were then cloned into the PCRscript
vector (Stratagene, La Jolla, Calif., USA) using the PCRscript Amp
cloning kit (Stratagene). Colonies containing inserts were picked
to 1-2 ml of LB broth with 100 .mu.g/ml ampicillin, 30-37.degree.
C., 16-24 hours, 230-300 rpm. Plasmid isolation was performed using
the Mo Bio miniplasmid prep kit (Mo Bio, Solano Beach, Calif.,
USA). This plasmid DNA was used as a PCR and sequencing template
and for restriction digest analysis.
[0061] The cloned PCR products were sequenced using the Big-Dye
sequencing kit from Applied Biosystems, (Foster City, Calif., USA)
and were analyzed using the ABI310 sequencer (Applied Biosystems,
Foster City, Calif., USA). The sequence of the inserts was used to
perform a TblastX search, using the protocol of Altschul, S. F, et
al., Mol. Biol. 215:403-410 (1990), of the non-redundant protein
database. Unique sequences having a significant similarity to known
cytochrome P450 proteins were retained. Using this approach, a
total of nine different P450 sequences were identified from
SC15847, seven from the genomic DNA template and two from the cDNA.
Two P450 sequences were found in common between the DNA and cDNA
templates. Of the fifty cDNA clones analyzed, two sequences were
predominant, with twenty clones each. These two genes were then
cloned from the genomic DNA.
[0062] The nucleic acid sequence of the genomic DNA was determined
using the Big-Dye sequencing system (Applied Biosystems) and
analyzed using an ABI310 sequencer. This sequence is depicted in
SEQ ID NO:1. An open reading frame coding for a protein of 404
amino acids and a predicted molecular weight of 44.7 kDa was found
within the cloned BglII fragment. The deduced amino acid sequence
of this polypeptide is depicted in SEQ ID NO: 2. The amino acid
sequence of this polypeptide was found to share 51% identity with
the NikF protein of Streptomyces tendae (Bruntner, C. et al, 1999,
Mol. Gen. Genet. 262: 102-114) and 48% identity with the Sca-2
protein of S. carbophilus (Watanabe, I. Et al, 1995, Gene 163:
81-85). Both of these enzymes belong to the cytochrome P450 family
105. The invariable cysteine found in the heme-binding domain of
all cytochrome P450 enzymes is found at residue 356. This gene for
epothilone B hydroxylase has been named ebh. The ATG start codon of
a putative ferredoxin gene of 64 amino acids is found nine
basepairs downstream from the stop codon of ebh. This enzyme was
found to share 50% identity with ferredoxin genes of S. griseoulus
(O'Keefe, D. P., et al, 1991, Biochemistry 30: 447-455) and S.
noursei (Brautaset, T., et al, 2000, Chem. Biol. 7: 395-403). The
nucleic acid sequence encoding this ferredoxin is depicted in SEQ
ID NO:3 and the amino acid sequence for this ferredoxin polypeptide
is depicted in SEQ ID NO:4.
[0063] The ebh gene sequence was also used to isolate variant
cytochrome P450 genes from other microorganisms. Exemplary variant
polynucleotides ebh43491, ebh14930, ebh53630, ebh53550, ebh39444,
ebh43333 and ebh35165 of the present invention and the species from
which they were isolated are depicted in Table 1 below. The nucleic
acid sequences for these variants are depicted in SEQ ID NO:36-42,
respectively.
2TABLE 1 Variant polynucleotides ATCC ID Species ebh gene
designation 43491 Amycolatopsis orientalis ebh43491 14930
Amycolatopsis orientalis ebh14930 53630 Amycolatopsis orientalis
ebh53630 53550 Amycolatopsis orientalis ebh53550 39444
Amycolatopsis orientalis ebh39444 43333 Amycolatopsis orientalis
ebh43333 35165 Amycolatopsis orientalis ebh35165
[0064] The amino acid sequences encoded by the exemplary variants
ebh43491, ebh14930, ebh53630, ebh53550, ebh39444, ebh43333 and
ebh35165 are depicted in SEQ ID NO:43-49, respectively. Table 2
provides a summary of the amino acid substitutions of these
exemplary variants.
3TABLE 2 Amino acid Substitutions Position ebh Substitution ebh
variant 100 Gly Ser ebh14930, ebh43333, ebh53550, ebh43491 101 Lys
Arg ebh14930 130 Ile Leu ebh14930 192 Ser Gln ebh14930 224 Ser Thr
ebh14930, ebh43333, ebh53550, ebh43491 285 Ile Vat ebh14930,
ebh43333, ebh53550, ebh43491 69 Ser Asn ebh43333 256 Val Ala
ebh43333, ebh53550, ebh43491 93 Ala Ser ebh53550 326 Asp Glu
ebh53550, ebh43491 333 Thr Ala ebh53550, ebh43491 133 Leu Met
ebh43491 398 His Arg ebh39444
[0065] Mutations were also introduced into the coding region of the
ebh gene to identify mutants with improved yield, and/or rate of
bioconversion and/or altered substrate specificity. Exemplary
mutant nucleic acid sequences of the present invention are depicted
in SEQ ID NO:30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74.
[0066] The nucleic acid sequence of SEQ ID NO:30 encodes a mutant
ebh25-1 which exhibits altered substrate specificity. Plasmid
pANT849ebh25-1 containing this mutant gene was deposited and
accepted by an International Depository Authority under the
provisions of the Budapest Treaty. The deposit was made on Nov.
______, 2002 to the American Type Culture Collection at 10801
University Boulevard in Manassas, Va. 20110-2209. The ATCC
Accession Number is ______. All restrictions upon public access to
this plasmid will be irrevocably removed upon granting of this
patent application. The Deposit will be maintained in a public
depository for a period of thirty years after the date of deposit
or five years after the last request for a sample or for the
enforceable life of the patent, whichever is longer. The
above-referenced plasmid was viable at the time of the deposit. The
deposit will be replaced if viable samples cannot be dispensed by
the depository.
[0067] This S. lividans transformant identified in the screening of
mutation 25 (primers NPB29-mut25f (SEQ ID NO:58) and NPB29-mut25r
(SEQ ID NO:59)) was found to produce a product with a different
HPLC elution time than epothilone B or epothilone F. A sample of
this unknown was analyzed by LC-MS and was found to have a
molecular weight of 523 (M.W.), consistent with a single
hydroxylation of epothilone B. Plasmid DNA was isolated from the S.
lividans culture and used as a template for PCR amplification using
primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29) (see
Example 17). The expected fragment was obtained and sequenced using
the Big-Dye sequencing system. The ebh25-1 mutant was found to have
two mutations resulting in changes in the amino acid sequence of
the protein, asparagine 195 is changed to serine and serine 294 is
changed to proline. The position targeted for mutation at codon 238
was found to have a two nucleotide change, which did not result in
a change of the amino acid sequence of the protein. The amino acid
sequence of the mutant polypeptide encoded by SEQ ID NO:30 is
depicted in SEQ ID NO:31.
[0068] The nucleic acid sequence of SEQ ID NO:32 encodes a mutant
ebh10-53, which exhibits improved bioconversion yield. This S.
lividans transformant identified in the screening of mutation 10
(primers NPB29-mut10f (SEQ ID NO:54) and NPB29-mut10r (SEQ ID
NO:55)) produced a greater yield of epothilone F. Plasmid DNA was
isolated from the S. lividans culture and used as a template for
PCR amplification using primers NPB29-6f (SEQ ID NO:28) and
NPB29-7r (SEQ ID NO:29)(see Example 16). The expected fragment was
obtained and sequenced using the Big-Dye sequencing system. The
ebh10-53 mutant was found to have two mutations resulting in
changes in the amino acid sequence of the protein, glutamic acid
231 is changed to arginine and phenylalanine 190 is changed to
tyrosine. The position 231 was the target of the mutagenesis, the
change at residue 190 is an inadvertent change that is an artifact
of the mutagenesis procedure. The amino acid sequence of the mutant
polypeptide encoded by SEQ ID NO:32 is depicted in SEQ ID
NO:33.
[0069] The nucleic acid sequence of SEQ ID NO:34 encodes a mutant
ebh24-16, which also exhibits improved bioconversion yield. This S.
lividans transformant, ebh24-16 identified in the screening of
mutation 24 (primers NPB29-mut24f (SEQ ID NO:56) and NPB29-mut24r
(SEQ ID NO:57) also produced a greater yield of epothilone F.
Plasmid DNA was isolated from the S. lividans culture and used as a
template for PCR amplification using primers NPB29-6f (SEQ ID
NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was
obtained and sequenced using the Big-Dye sequencing system. The
ebh24-16 mutant was found to have two mutations resulting in
changes in the amino acid sequence of the protein, phenylalanine
237 is changed to alanine and isoleucine 92 is changed to valine.
The position 237 was the target of the mutagenesis, the change at
residue 92 is an inadvertent change that is an artifact of the
mutagenesis procedure. The amino acid sequence of the mutant
polypeptide encoded by SEQ ID NO:34 is depicted in SEQ ID
NO:35.
[0070] The nucleic acid sequence of SEQ ID NO:60 encodes a mutant
ebh24-16d8, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebh24-16d8 identified in the screening of
mutation 59 (primer NPB29mut59 (SEQ ID NO:77)) also produced a
greater yield of epothilone F. Plasmid DNA was isolated from the S.
rimosus culture and used as a template for PCR amplification using
primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The
expected fragment was obtained and sequenced using the Big-Dye
sequencing system. The ebh24-16d8 mutant was found to have one
mutation resulting in a change in the amino acid sequence of the
protein, arginine 67 is changed to glutamine. This change is an
artifact of the mutagenesis procedure. The amino acid sequence of
the mutant polypeptide encoded by SEQ ID NO:60 is SEQ ID NO:61.
[0071] The nucleic acid sequence of SEQ ID NO:62 encodes a mutant
ebh24-16c11, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebh24-16c11 identified in the screening of
mutation 59 (primer NPB29mut59 (SEQ ID NO:77)) also produced a
greater yield of epothilone F. Plasmid DNA was isolated from the S.
rimosus culture and used as a template for PCR amplification using
primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The
expected fragment was obtained and sequenced using the Big-Dye
sequencing system. The ebh24-16c11 mutant was found to have two
additional mutations resulting in changes in the amino acid
sequence of the protein, alanine 93 is changed to glycine and
isoleucine 365 is changed to threonine. The position 93 is the
target of the mutagenesis, the change at 365 is an artifact of the
mutagenesis procedure. The amino acid sequence of the mutant
polypeptide encoded by SEQ ID NO:62 is depicted in SEQ ID
NO:63.
[0072] The nucleic acid sequence of SEQ ID NO:64 encodes a mutant
ebh24-16-16, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebh24-16-16 identified in the screening of
random mutants of ebh24-16 also produced a greater yield of
epothilone F. Plasmid DNA was isolated from the S. rimosus culture
and used as a template for PCR amplification using primers NPB29-6f
(SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment
was obtained and sequenced using the Big-Dye sequencing system. The
ebh24-16-16 mutant was found to have one additional mutation
resulting in changes in the amino acid sequence of the protein,
valine 106 is changed to alanine. The amino acid sequence of the
mutant polypeptide encoded by SEQ ID NO:64 is depicted in SEQ ID
NO:65.
[0073] The nucleic acid sequence of SEQ ID NO:66 encodes a mutant
ebh24-16-74, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebh24-16-74 identified in the screening of
random mutants of ebh24-16 also produced a greater yield of
epothilone F. Plasmid DNA was isolated from the S. rimosus culture
and used as a template for PCR amplification using primers NPB29-6f
(SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment
was obtained and sequenced using the Big-Dye sequencing system. The
ebh24-16-74 mutant was found to have one additional mutation
resulting in changes in the amino acid sequence of the protein,
arginine 88 is changed to histidine. The amino acid sequence of the
mutant polypeptide encoded by SEQ ID NO:66 is SEQ ID NO:67.
[0074] The nucleic acid sequence of SEQ ID NO:68 encodes a mutant
ebh24-M18, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebhM-18 identified in the screening of
random mutants of ebh also produced a greater yield of epothilone
F. Plasmid DNA was isolated from the S. rimosus culture and used as
a template for PCR amplification using primers NPB29-6f (SEQ ID
NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was
obtained and sequenced using the Big-Dye sequencing system. The
ebhM-18 mutant was found to have two mutations resulting in changes
in the amino acid sequence of the protein, glutamic acid 31 is
changed to lysine and methionine 176 is changed to valine. The
amino acid sequence of the mutant polypeptide encoded by SEQ ID
NO:68 is depicted in SEQ ID NO:69.
[0075] The nucleic acid sequence of SEQ ID NO:72 encodes a mutant
ebh24-16g8, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebh24-16g8 identified in the screening of
mutation 50 (primer NPB29mut50 (SEQ ID NO:78)) also produced a
greater yield of epothilone F. Plasmid DNA was isolated from the S.
rimosus culture and used as a template for PCR amplification using
primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The
expected fragment was obtained and sequenced using the Big-Dye
sequencing system. The ebh24-16g8 mutant was found to have two
additional mutations resulting in changes in the amino acid
sequence of the protein, methionine 176 is changed to alanine and
isoleucine 130 is changed to threonine. The position 176 is the
target of the mutagenesis, the change at 130 is an artifact of the
mutagenesis procedure. The amino acid sequence of the mutant
polypeptide encoded by SEQ ID NO:72 is depicted in SEQ ID
NO:73.
[0076] The nucleic acid sequence of SEQ ID NO:74 encodes a mutant
ebh24-16b9, which also exhibits improved bioconversion yield. This
S. rimosus transformant, ebh24-16b9 identified in the screening of
mutation 50 (primer NPB29mut50 (SEQ ID NO:78)) also produced a
greater yield of epothilone F. Plasmid DNA was isolated from the S.
rimosus culture and used as a template for PCR amplification using
primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The
expected fragment was obtained and sequenced using the Big-Dye
sequencing system. The ebh24-16b9 mutant was found to have two
additional mutations resulting in changes in the amino acid
sequence of the protein, methionine 176 is changed to serine and
alanine 140 is changed to threonine. The position 176 is the target
of the mutagenesis, the change at 140 is an artifact of the
mutagenesis procedure. The amino acid sequence of the mutant
polypeptide encoded by SEQ ID NO:74 is depicted in SEQ ID
NO:75.
[0077] A mixture composed of the plasmids pANT849ebh-24-16,
pANT849ebh-10-53, pANT849ebh-24-16d8, pANT849ebh-24-16c11,
pANT849ebh-24-16-16, pant849ebh-24-16-74, pANT849ebh-24-16b9,
pANT849ebh-M18 and pANT849ebh-24-16g8 for these nine mutant genes
was deposited and accepted by an International Depository Authority
under the provisions of the Budapest Treaty. The deposit was made
on Nov. ______, 2002 to the American Type Culture Collection at
10801 University Boulevard in Manassas, Va. 20110-2209. The ATCC
Accession Number is ______. All restrictions upon public access to
this mixture of plasmids will be irrevocably removed upon granting
of this patent application. The deposit will be maintained in a
public depository for a period of thirty years after the date of
deposit or five years after the last request for a sample or for
the enforceable life of the patent, whichever is longer. The
above-referenced mixture of plasmids was viable at the time of the
deposit. The deposit will be replaced if viable samples cannot be
dispensed by the depository.
[0078] Thus, in accordance with another aspect of the present
invention, there are provided isolated polypeptides of epothilone B
hydroxylase and variants and mutants thereof and isolated
polypeptides of ferredoxin or variants thereof. In one embodiment
of the present invention, by "polypeptide" it is meant to include
the amino acid sequence of SEQ ID NO: 2, and fragments or variants,
which retain essentially the same biological activity and/or
function as this epothilone B hydroxylase. In another embodiment of
the present invention, by "polypeptide" it is meant to include the
amino acid sequence of SEQ ID NO:4, and fragments and/or variants,
which retain essentially the same biological activity and/or
function as this ferredoxin.
[0079] By "variants" as used herein it is meant to include
polypeptides with amino acid sequences with conservative amino acid
substitutions as compared to SEQ ID NO: 2 or 4 which are
demonstrated to exhibit similar biological activity and/or function
to SEQ ID NO:2 or 4. By "conservative amino acid substitutions" it
is meant to include replacement, one for another, of the aliphatic
amino acids such as Ala, Val, Leu and Ile, the hydroxyl residues
Ser and Thr, the acidic residues Asp and Glu, and the amide
residues Asn and Gln. Exemplary variant amino acid sequences of the
present invention are depicted in SEQ ID NO:43-49 and the amino
acid substitutions of these exemplary variants are described in
Table 2, supra.
[0080] By "mutants" as used herein it is meant to include
polypeptides encoded by nucleic acid sequences with one or more
point mutations, or deletions or additions of nucleic acids as
compared to SEQ ID NO: 1 or 3, but which still have similar
activity to the polypeptides encoded by SEQ ID NO: 1 or 3. In a
preferred embodiment, mutations are made to the nucleic acid that
alter the substrate specificity and/or yield from the polypeptide
encoded thereby. A preferred region of mutation with respect to the
epothilone B hydroxylase gene is that region of the nucleic acid
sequence coding for the approximately 113 amino acid residues
comprising the active site of the enzyme. Also preferred are
mutants with at least one amino acid substitution at amino acid
position GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140,
MET176, PHE190, GLU 231, SER294, PHE237, or ILE365 of SEQ ID NO:1
Exemplary mutants ebh25-1, ebh10-53, ebh24-16, ebh24-16d8,
ebh24-16c11, ebh24-16-16, ebh24-16-74, ebh24-16g8, ebh24-16b9 and
the nucleic acid sequences encoding such mutants of the present
invention are depicted in SEQ ID NO:31, 33, 35, 61, 63, 65, 67, 69,
71, 73 and 75, and SEQ ID NO:30, 32, 34, 60, 62, 64, 66, 68, 70, 72
and 74, respectively.
[0081] A 3-dimensional model of epothilone B hydroxylase has also
been constructed in accordance with general teachings of Greer et
al. (Comparative modeling of homologous proteins. Methods In
Enzymology 202239-52, 1991), Lesk et al. (Homology Modeling:
Inferences from Tables of Aligned Sequences. Curr. Op. Struc. Biol.
(2) 242-247, 1992), and Cardozo et al. (Homology modeling by the
ICM method. Proteins 23, 403-14, 1995) on the basis of the known
structure of a homologous protein EryF (PDB Code 1KIN chain A).
Homology between these sequences is 34%. Alignment of the sequences
of epothilone B hydroxylase (SEQ ID NO:2) and EryF (PDB Code 1KIN
chain A; SEQ ID NO:76) is depicted in FIG. 3. A homology model of
epothilone B hydroxylase based upon sequence alignment with EryF is
depicted in FIG. 4.
[0082] An energy plot of the epothilone B hydroxylase model
relative to EryF (PDB code 1JIN) was also prepared and is depicted
in FIG. 5. An averaging window size of 51 residues was used at a
given residue position to calculate the average of the energies of
the 51 residues in the sequence that lie with the given residue at
the central position. As shown in FIG. 5, all energies along the
sequence lie below zero thus indicating that the modeled structure
as set forth in FIG. 4 and Appendix 1 is reasonable.
[0083] The three-dimensional structure represented in the homology
model of epothilone B hydroxylase of FIG. 4 is defined by a set of
structure coordinates as set forth in Appendix 1. The term
"structure coordinates" refers to Cartesian coordinates generated
from the building of a homology model. As will be understood by
those of skill in the art, however, a set of structure coordinates
for a protein is a relative set of points that define a shape in
three dimensions. Thus, it is possible that an entirely different
set of coordinates could define a similar or identical shape.
Moreover, slight variations in the individual coordinates, as
emanate from generation of similar homology models using different
alignment templates and/or using different methods in generating
the homology model, will have minor effects on the overall shape.
Variations in coordinates may also be generated because of
mathematical manipulations of the structure coordinates. For
example, the structure coordinates set forth in Appendix 1 could be
manipulated by fractionalization of the structure coordinates;
integer additions or subtractions to sets of the structure
coordinates, inversion of the structure coordinates or any
combination of the above.
[0084] Various computational analyses are therefore necessary to
determine whether a molecule or a portion thereof is sufficiently
similar to all or parts of epothilone B hydroxylase described above
as to be considered the same. Such analyses may be carried out in
current software applications, such as SYBYL version 6.7 or
INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version
2000 and as described in the accompanying User's Guides.
[0085] For example, the superimposition tool in the program SYBYL
allows comparisons to be made between different structures and
different conformations of the same structure. The procedure used
in SYBYL to compare structures is divided into four steps: 1) load
the structures to be compared; 2) define the atom equivalencies in
these structures; 3) perform a fitting operation; and 4) analyze
the results. Each structure is identified by a name. One structure
is identified as the target (i.e., the fixed structure); the second
structure (i.e., moving structure) is identified as the source
structure. Since atom equivalency within SYBYL is defined by user
input, for the purpose of this aspect of the present invention
equivalent atoms are defined as protein backbone atoms (N,
C.alpha., C and O) for all conserved residues between the two
structures being compared. Further, only rigid fitting operations
are considered. When a rigid fitting method is used, the working
structure is translated and rotated to obtain an optimum fit with
the target structure. The fitting operation uses an algorithm that
computes the optimum translation and rotation to be applied to the
moving structure, such that the root mean square difference of the
fit over the specified pairs of equivalent atoms is an absolute
minimum. This number, given in angstroms, is reported by SYBYL.
[0086] For the purposes of the present invention, any homology
model of epothilone B hydroxylase that has a root mean square
deviation of conserved residue backbone atoms (N, C.alpha., C, O)
of less than about 4.0 .ANG. when superimposed on the corresponding
backbone atoms described by structure coordinates listed in
Appendix 1 are considered identical. More preferably, the root mean
square deviation is less than about 3.0 .ANG.. More preferably the
root mean square deviation is less than about 2.0 .ANG..
[0087] For the purpose of this invention, any homology model of
epothilone B hydroxylase that has a root mean square deviation of
conserved residue backbone atoms (N, C.alpha., C, O) of less than
about 2.0 .ANG. when superimposed on the corresponding backbone
atoms described by structure coordinates listed in Appendix 1 are
considered identical. More preferably, the root mean square
deviation is less than about 1.0 .ANG..
[0088] In another embodiment of the present invention, structural
models wherein backbone atoms have been substituted with other
elements which when superimposed on the corresponding backbone
atoms have low root mean square deviations are considered to be
identical. For example, an homology model where the original
backbone carbon, and/or nitrogen and/or oxygen atoms are replaced
with other elements having a root mean square deviation of about
4.0 .ANG., more preferably about 3.0 .ANG., even more preferably
less than about 2 .ANG., when superimposed on the corresponding
backbone atoms described by structure coordinates listed in
Appendix 1 is considered identical.
[0089] The term "root mean square deviation" means the square root
of the arithmetic mean of the squares of the deviations from the
mean. It is a way to express the deviation or variation from a
trend or object. For purposes of this invention, the "root mean
square deviation" defines the variation in the backbone of a
protein from the relevant portion of the backbone of the epothilone
B hydroxylase portion of the complex as defined by the structure
coordinates described herein.
[0090] The present invention as embodied by the homology model
enables the structure-based design of additional mutants of
epothilone B hydroxylase. For example, using the homology model of
the present invention, residues lying within 10 .ANG. of the
binding site of epothilone B hydroxylase have now been defined.
These residues include LEU39, GLN43, ALA45, MET57, LEU58, HIS62,
PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75,
VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, LYS85, PRO86, PHE87,
ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99,
ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174,
ARG175, MET176, LEU177, SER178, ARG179, ARG186, PHE190, LEU193,
VAL233, GLY234, LEU235, ALA236, PHE237, LEU238, LEU239, LEU240,
ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248,
ASN249, MET250, LEU283, THR287, ILE288, ALA289, GLU290, THR291,
ALA292, THR293, SER294, ARG295, PHE296, ALA297, THR298, GLU312,
GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347,
PHE348, VAL350, HIS351, GLN352, CYS353, LEU354, GLY355, GLN356,
LEU358, ALA359, GLU362, LYS389, ASP391, SER392,THR393, ILE394 and
TYR395 as set forth in Appendix 1. Mutants with mutations at one or
more of these positions are expected to exhibit altered biological
function and/or specificity and thus comprise another embodiment of
preferred mutants of the present invention. Another embodiment of
preferred mutants are molecules that have a root mean square
deviation from the backbone atoms of said epothilone B hydroxylase
of not more than about 4.0 .ANG..
[0091] The structure coordinates of an epothilone B hydroxylase
homology model or portions thereof are stored in a machine-readable
storage medium. Such data may be used for a variety of purposes,
such as drug discovery.
[0092] Accordingly, another aspect of the present invention relates
to machine-readable data storage medium comprising a data storage
material encoded with the structure coordinates set forth in
Appendix 1.
[0093] The three-dimensional model structure of epothilone B
hydroxylase can also be used to identify modulators of biological
function and potential substrates of the enzyme. Various methods or
combinations thereof can be used to identify such modulators.
[0094] For example, a test compound can be modeled that fits
spatially into a binding site in epothilone B hydroxylase,
according to Appendix 1. Structure coordinates of amino acids
within 10 .ANG. of the binding region of epothilone B hydroxylase
defined by amino acids LEU39, GLN43, ALA45, MET57, LEU58, HIS62,
PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75,
VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, LYS85, PRO86, PHE87,
ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99,
ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174,
ARG175, MET176, LEU177, SER178, ARG179, ARG186, PHE190, LEU193,
VAL233, GLY234, LEU235, ALA236, PHE237, LEU238, LEU239, LEU240,
ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248,
ASN249, MET250, LEU283, THR287, ILE288, ALA289, GLU290, THR291,
ALA292, THR293, SER294, ARG295, PHE296, ALA297, THR298, GLU312,
GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347,
PHE348, VAL350, HIS351, GLN352, CYS353, LEU354, GLY355, GLN356,
LEU358, ALA359, GLU362, LYS389, ASP391, SER392,THR393, ILE394 and
TYR395, and the coordinated heme group, HEM1 can also be used to
identify desirable structural and chemical features of such
modulators. Identified structural or chemical features can then be
employed to design or select compounds as potential epothilone B
hydroxylase ligands. By structural and chemical features it is
meant to include, but is not limited to, covalent bonding, van der
Waals interactions, hydrogen bonding interactions, charge
interaction, hydrophobic bonding interaction, and dipole
interaction. Compounds identified as potential epothilone B
hydroxylase ligands can then be synthesized and screened in an
assay characterized by binding of a test compound to epothilone B
hydroxylase, or in characterizing the ability of epothilone B
hydroxylase to modulate a protease target in the presence of a
small molecule. Examples of assays useful in screening of potential
epothilone B hydroxylase ligands include, but are not limited to,
screening in silico, in vitro assays and high throughput
assays.
[0095] As will be understood by those of skill in the art upon this
disclosure, other structure-based design methods can be used.
Various computational structure-based design methods have been
disclosed in the art. For example, a number of computer modeling
systems are available in which the sequence of epothilone B
hydroxylase and the epothilone B hydroxylase structure (i.e.,
atomic coordinates of epothilone B hydroxylase as provided in
Appendix 1 and/or the atomic coordinates within 10 .ANG. of the
binding region as provided above) can be input. This computer
system then generates the structural details of one or more these
regions in which a potential epothilone B hydroxylase modulator bi
modulator binds so that complementary structural details of the
potential modulators can be determined. Design in these modeling
systems is generally based upon the compound being capable of
physically and structurally associating with hydroxylase. In
addition, the compound must be able to assume a conformation that
allows it to associate with epothilone B hydroxylase. Some modeling
systems estimate the potential inhibitory or binding effect of a
potential epothilone B hydroxylase substrate or modulator prior to
actual synthesis and testing.
[0096] Methods for screening chemical entities or fragments for
their ability to associate with a given protein target are also
well known. Often these methods begin by visual inspection of the
binding site on the computer screen. Selected fragments or chemical
entities are then positioned in a binding region of epothilone B
hydroxylase. Docking is accomplished using software such as
INSIGHTII, QUANTA and SYBYL, following by energy minimization and
molecular dynamics with standard molecular mechanic force fields
such as, MMFF, CHARMM and AMBER. Examples of computer programs
which assist in the selection of chemical fragment or chemical
entities useful in the present invention include, but are not
limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and
DOCK (Kuntz et al. 1982).
[0097] Upon selection of preferred chemical entities or fragments,
their relationship to each other and epothilone B hydroxylase can
be visualized and then assembled into a single potential modulator.
Programs useful in assembling the individual chemical entities
include, but are not limited to CAVEAT (Bartlett et al. 1989) and
3D Database systems (Martin 1992).
[0098] Alternatively, compounds may be designed de novo using
either an empty active site or optionally including some portion of
a known inhibitor. Methods of this type of design include, but are
not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Inc., St.
Louis Mo.).
[0099] Programs such as DOCK (Kuntz et al. 1982) can be used with
the atomic coordinates from the homology model to identify
potential ligands from databases or virtual databases which
potentially bind the in the active site binding region which may
therefore be suitable candidates for synthesis and testing.
[0100] Also provided in the present invention are vectors
comprising polynucleotides of the present invention and host cells
which are genetically engineered with vectors of the present
invention to produce epothilone B hydroxylase or active fragments
and variants or mutants of this enzyme and/or ferredoxin or active
fragments thereof. Generally, any vector suitable to maintain,
propagate or express polynucleotides to produce these polypeptides
in the host cell may be used for expression in this regard. In
accordance with this aspect of the invention the vector may be, for
example, a plasmid vector, a single- or double-stranded phage
vector, or a single- or double-stranded RNA or DNA viral vector.
Vectors may be extra-chromosomal or designed for integration into
the host chromosome. Such vectors include, but are not limited to,
chromosomal, episomal and virus-derived vectors e.g., vectors
derived from bacterial plasmids, bacteriophages, yeast episomes,
yeast chromosomal elements, and viruses such as baculoviruses,
papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox
viruses, pseudorabies viruses and retroviruses, and vectors derived
from combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements, cosmids and phagemids.
[0101] Useful expression vectors for prokaryotic hosts include, but
are not limited to, bacterial plasmids, such as those from E. coli,
Bacillus or Streptomyces, including pBluescript, pGEX-2T, pUC
vectors, pET vectors, ColE1, pCR1, pBR322, pMB9, pCW, pBMS200,
pBMS2020, PIJ101, PIJ702, pANT849, pOJ260, pOJ446, pSET152,
pKC1139, pKC1218, pFD666 and their derivatives, wider host range
plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives
of phage lambda, e.g., NM989, .lambda.GT10 and .lambda.GT11, and
other phages, e.g., M13 and filamentous single stranded phage
DNA.
[0102] Vectors of the present invention for use in yeast will
typically contain an origin of replication suitable for use in
yeast and a selectable marker that is functional in yeast. Examples
of yeast vectors useful in the present invention include, but are
not limited to, Yeast Integrating plasmids (e.g., YIp5) and Yeast
Replicating plasmids (the YRp and YEp series plasmids), Yeast
Centromere plasmids (the YCp series plasmids), Yeast Artificial
Chromosomes (YACs) which are based on yeast linear plasmids,
denoted YLp, pGPD-2, 2.mu. plasmids and derivatives thereof, and
improved shuttle vectors such as those described in Gietz et al.,
Gene, 74: 527-34 (1988) (YIplac, YEplac and YCplac).
[0103] Mammalian vectors useful for recombinant expression may
include a viral origin, such as the SV40 origin (for replication in
cell lines expressing the large T-antigen, such as COS1 and COS7
cells), the papillomavirus origin, or the EBV origin for long term
episomal replication (for use, e.g., in 293-EBNA cells, which
constitutively express the EBV EBNA-1 gene product and adenovirus
E1A). Expression in mammalian cells can be achieved using a variety
of plasmids, including, but not limited to, pSV2, pBC12BI, and
p91023, pCDNA vectors as well as lytic virus vectors (e.g.,
vaccinia virus, adeno virus, and baculovirus), episomal virus
vectors (e.g., bovine papillomavirus), and retroviral vectors
(e.g., murine retroviruses). Useful vectors for insect cells
include baculoviral vectors and pVL941.
[0104] Selection of an appropriate promoter to direct mRNA
transcription and construction of expression vectors are well
known. In general, however, expression constructs will contain
sites for transcription initiation and termination, and, in the
transcribed region, a ribosome binding site for translation. The
coding portion of the mature transcripts expressed by the
constructs will include a translation initiating codon at the
beginning and a termination codon appropriately positioned at the
end of the polypeptide to be translated.
[0105] Examples of useful promoters for prokaryotes include, but
are not limited to phage promoters such as phage lambda pL
promoter, the trc promoter, a hybrid derived from the trp and lac
promoters, the bacteriophage T7 promoter, the TAC or TRC system,
the major operator and promoter regions of phage lambda, the
control regions of fd coat protein, snpA promoter, melC promotor,
ermE* promoter or the araBAD operon. Examples of useful promoters
for yeast include, but are not limited to, the CYC1 promoter, the
GAL1 promoter, the GAL10 promoter, ADH1 promoter, the promoters of
the yeast .alpha.-mating system, and the GPD promoter. Examples of
promoters routinely used in mammalian expression vectors include,
but are not limited to, the CMV immediate early promoter, the HSV
thymidine kinase promoter, the early and late SV40 promoters, the
promoters of retroviral LTRs, such as those of the Rous Sarcoma
Virus(RSV), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0106] Vectors comprising the polynucleotides can be introduced
into host cells using any number of well known techniques including
infection, transduction, transfection, transvection and
transformation. The polynucleotides may be introduced into a host
alone or with additional polynucleotides encoding, for example, a
selectable marker or ferredoxin reductase. In a preferred
embodiment of the present invention the polynucleotide for
epothilone B hydroxylase and ferredoxin are introduced into the
host cell. Host cells for the various expression constructs are
well known, and those of skill can routinely select a host cell for
expressing the epothilone B hydroxylase and/or ferredoxin in
accordance with this aspect of the present invention. Examples of
mammalian expression systems useful in the present invention
include, but are not limited to, the C127, 3T3, CHO, HeLa, human
kidney 293 and BHK cell lines, and the COS-7 line of monkey kidney
fibroblasts.
[0107] Alternatively, as exemplified herein, epothilone B
hydroxylase and ferredoxin can be expressed recombinantly in
microorganisms.
[0108] Accordingly, another aspect of the present invention relates
to recombinantly produced microorganisms which express epothilone B
hydroxylase alone or in conjunction with the ferredoxin and which
are capable of hydroxylating a compound, and in particular an
epothilone, having a terminal alkyl group to produce ones having a
terminal hydroxyalkyl group. The recombinantly produced
microorganisms are produced by transforming cells such as bacterial
cells with a plasmid comprising a nucleic acid sequence encoding
epothilone B hydroxylase. In a preferred embodiment, the cells are
transformed with a plasmid comprising a nucleic acid encoding
epothilone B hydroxylase or mutants or variants thereof as well as
the nucleic acid sequence encoding ferredoxin located downstream of
the epothilone B hydroxylase gene. Examples of microorganisms which
can be transformed with these plasmids to produce the recombinant
microorganisms of the present invention include, but are not
limited, Escherichia coli, Bacillus megaterium, Amycolatopsis
orientalis, Sorangium cellulosum, Rhodococcus erythropolis, and
Streptomyces species such as Streptomyces lividans, Streptomyces
virginiae, Streptomyces venezuelae, Streptomyces albus,
Streptomyces coelicolor, Streptomyces rimosus and Streptomyces
griseus.
[0109] The recombinantly produced microorganisms of the present
invention are useful in microbial processes or methods for
production of compounds, and in particular epothilones, containing
a terminal hydroxyalkyl group. In general, the hydroxyalkyl-bearing
product can be produced by culturing the recombinantly produced
microorganism or enzyme derived therefrom, capable of selectively
hydroxylating a terminal carbon or alkyl, in the presence of a
suitable substrate in an aqueous nutrient medium containing sources
of assimilable carbon and nitrogen, under submerged aerobic
conditions.
[0110] Suitable epothilones employed as substrate for the method of
the present invention may be any such compound having a terminal
carbon or terminal alkyl group capable of undergoing the enzymatic
hydroxylation of the present invention. The starting material, or
substrate, can be isolated from natural sources, such as Sorangium
cellulosum, or they can be synthetically formed epothilones. Other
substrates having a terminal carbon or terminal alkyl group capable
of undergoing an enzymatic hydroxylation can be employed by the
methods herein. For example, compactin can be used as a substrate,
which upon hydroxylation forms the compound pravastatin. Methods
for hydroxylating compactin to pravastatin via an Actinomadura
strain are set forth in U.S. Pat. No. 5,942,423 and U.S. Pat. No.
6,274,360.
[0111] For example, using the recombinant microorganisms of the
present invention at least one epothilone can be prepared as
described in WO 00/39276, U.S. Ser. No. 09/468,854, filed Dec. 21,
1999, the text of which is incorporated herein as if set forth at
length. An epothilone of the following Formula I
HO--CH.sub.2--(A.sub.1).sub.n-(Q).sub.m--(A.sub.2).sub.o--E (I)
[0112] where
[0113] A.sub.1 and A.sub.2 are independently selected from the
group of optionally substituted C.sub.1-C.sub.3 alkyl and
alkenyl;
[0114] Q is an optionally substituted ring system containing one to
three rings and at least one carbon to carbon double bond in at
least one ring;
[0115] n, m, and o are integers selected from the group consisting
of zero and 1, where at least one of m or n or o is 1; and
[0116] E is an epothilone core; can be prepared.
[0117] This method comprises the steps of contacting at least one
epothilone of the following formula II
CH.sub.3--(A.sub.1).sub.n--(Q).sub.m--(A.sub.2).sub.o--E (II)
[0118] where A.sub.1, Q, A.sub.2, E, n, m, and o are defined as
above;
[0119] with a recombinantly produced microorganism, or an enzyme
derived therefrom, which is capable of selectively catalyzing the
hydroxylation of formula II, and effecting said hydroxylation.
[0120] In a preferred embodiment, the starting material is
epothilone B. Epothilone B can be obtained from the fermentation of
Sorangium cellulosum So ce90, as described in DE 41 38 042 and WO
93/10121. The strain has been deposited at the Deutsche Sammlung
von Mikroorganismen (German Collection of Microorganisms) (DSM)
under No. 6773. The process of fermentation is also described in
Hofle, G., et al., Angew. Chem. Int. Ed. Engl., Vol 35, No. 13/14,
1567-1569 (1996). Epothilone B can also be obtained by chemical
means, such as those disclosed by Meng, D., et al.,J. Am. Chem.
Soc., Vol. 119, No. 42, 10073-10092 (1996); Nicolaou, K., et al.,
J. Am. Chem. Soc., Vol. 119, No. 34, 7974-7991 (1997) and Schinzer,
D., et al., Chem. Eur. J., Vol. 5, No. 9, 2483-2491 (1999).
[0121] Growth of the recombinantly produced microorganism selected
for use in the process may be achieved by one of ordinary skill in
the art by the use of appropriate nutrient medium. Appropriate
media for the growing of the recombinantly produced microorganisms
include those that provide nutrients necessary for the growth of
microbial cells. See, for example, T. Nagodawithana and J. M.
Wasileski, Chapter 2: "Media Design for Industrial Fermentations,"
Nutritional Requirements of Commercially Important Microorganism,
edited by T. W. Nagodawithana and G. Reed, Esteekay Associates,
Inc., Milwaukee, Wis., 18-45 (1998); T. L. Miller and B. W.
Churchill, Chapter 10: "Substrates for Large-Scale Fermentations,"
Manual of Industrial Microbiology and Biotechnology, edited by A.
L. Demain and N. A. Solomon, American Society for Microbiology,
Washington, D.C., 122-136 (1986). A typical medium for growth
includes necessary carbon sources, nitrogen sources, and trace
elements. Inducers may also be added to the medium. The term
inducer as used herein, includes any compound enhancing formation
of the desired enzymatic activity within the recombinantly produced
microbial cell. Typical inducers as used herein may include
solvents used to dissolve substrates, such as dimethyl sulfoxide,
dimethyl formamide, dioxane, ethanol and acetone. Further, some
substrates, such as epothilone B, may also be considered to be
inducers.
[0122] Carbon sources may include sugars such as glucose, fructose,
galactose, maltose, sucrose, mannitol, sorbital, glycerol starch
and the like; organic acids such as sodium acetate, sodium citrate,
and the like; and alcohols such as ethanol, propanol and the like.
Preferred carbon sources include, but are not limited to, glucose,
fructose, sucrose, glycerol and starch.
[0123] Nitrogen sources may include an N-Z amine A, corn steeped
liquor, soybean meal, beef extract, yeast extract, tryptone,
peptone, cottonseed meal, peanut meal, amino acids such as sodium
glutamate and the like, sodium nitrate, ammonium sulfate and the
like.
[0124] Trace elements may include magnesium, manganese, calcium,
cobalt, nickel, iron, sodium and potassium salts. Phosphates may
also be added in trace or preferably, greater than trace
amounts.
[0125] The medium employed for the fermentation may include more
than one carbon or nitrogen source or other nutrient.
[0126] For growth of the recombinantly produced microorganisms
and/or hydroxylation according to the method of the present
invention, the pH of the medium is preferably from about 5 to about
8 and the temperature is from about 14.degree. C. to about
37.degree. C., preferably the temperature is 28.degree. C. The
duration of the reaction is 1 to 100 hours, preferably 8 to 72
hours.
[0127] The medium is incubated for a period of time necessary to
complete the biotransformation as monitored by high performance
liquid chromatography (HPLC). Typically, the period of time needed
to complete the transformation is twelve to one hundred hours and
preferably about 72 hours after the addition of the substrate. The
medium is placed on a rotary shaker (New Brunswick Scientific
Innova 5000) operating at 150 to 300 rpm and preferably about 250
rpm with a throw of 2 inches.
[0128] The hydroxyalkyl-bearing product can be recovered from the
fermentation broth by conventional means that are commonly used for
the recovery of other known biologically active substances.
Examples of such recovery means include, but are not limited to,
isolation and purification by extraction with a conventional
solvent, such as ethyl acetate and the like; by pH adjustment; by
treatment with a conventional resin, for example, by treatment with
an anion or cation exchange resin or a non-ionic adsorption resin;
by treatment with a conventional adsorbent, for example, by
distillation, by crystallization; or by recrystallization, and the
like.
[0129] The extract obtained above from the biotransformation
reaction mixture can be further isolated and purified by column
chromatography and analytical thin layer chromatography.
[0130] The ability of a recombinantly produced microorganism of the
present invention to biotransform an epothilone having a terminal
alkyl group to an epothilone having a terminal hydroxyalkyl group
was demonstrated. In these experiments, a culture comprising a
Streptomyces lividans clone containing a plasmid with the ebh gene
as described in more detail in Example 11 was incubated with an
epothilone B suspension for 3 days at 30.degree. with agitation. A
sample of the incubate was extracted with an equal volume of 25%
methanol: 75% n-butanol, vortexed and allowed to settle for 5
minutes. Two hundred .mu.l of the organic phase was transferred to
an HPLC vial and analyzed by HPLC/MS (Example 12). A product peak
of epothilone F eluted at a retention time of 15.9 minutes and had
a protonated molecular weight of 524. The epothilone B substrate
eluted at 19.0 minutes and had a protonated molecular weight of
508. The peak retention times and molecular weights were confirmed
using known standards.
[0131] Rates of biotransformation of epothilone B by cells
expressing ebh were also compared to rates of biotransformation by
ebh mutants. Cells expressing ebh comprised a frozen spore
preparation of. S. lividans (pANT849-ebh). Cells expressing mutants
comprises frozen spore preparations of S. lividans
(pANT849-ebh10-53) and S. lividans (pANT849-ebh24-16). A frozen
spore preparation of S. lividans TK24 was used as the control. The
cells were pre-incubated for several days at 30.degree. C.
Following this pre-incubation, epothilone B in 100% EtOH was added
to each culture to a final concentration of 0.05% weight/volume.
Samples were then taken at 0, 24, 48 and 72 hours with the
exception of the S. lividans (pANT849-ebh24-16) culture, in which
the epothilone B had been completely converted to epothilone F at
48 hours. The samples were analyzed by HPLC. The results are
calculated as a percentage of the epothilone B at time 0 hours.
4 Epothilone B: Time pANT849- pANT849- (hours) TK24 pANT849-ebh
ebh10-53 ebh24-16 0 100% 100% 100% 100% 24 99% 78% 69% 56% 48 87%
19% 39% 0% 72 87% 0% 3% --
[0132]
5 Epothilone F: Time pANT849- pANT849- (hours) TK24 pANT849-ebh
ebh10-53 ebh24-16 0 0% 0% 0% 0% 24 0% 4% 9% 23% 48 0% 21% 29% 52%
72 0% 14% 41% --
[0133] The ability of cells expressing ebh to biotransform
compactin to pravastatin was also examined. In these experiments,
frozen spore preparations of S. lividans (pANT849) or S. lividans
(pANT849-ebh) were grown for several days at 30.degree. C.
Following the pre-incubation, an aliquot of each cell culture was
transferred to a polypropylene culture tube, compactin was added to
each culture tube, and the tubes were incubated for 24 hours,
30.degree. C., 250 rpm. An aliquot of the culture broth was then
extracted and compactin and pravastatin values relative to the
control S. lividans (pANT849) culture were measured via HPLC.
[0134] Compactin and pravastatin as a percentage of starting
compactin concentration:
6 S. lividans (pANT849) S. lividans (pANT849-ebh) Compactin 36% 11%
Pravastatin 11% 53%
[0135] As discussed supra, mutant ebh25-1 (SEQ ID NO:30) exhibits
altered substrate specificity and biotransformation of epothilone B
by this mutant resulted in a product with a different HPLC elution
time than epothilone B or epothilone F. A sample of this unknown
was analyzed by LC-MS and was found to have a molecular weight of
523 (M.W.), consistent with a single hydroxylation of epothilone B.
The structure of the biotransformation product was determined as
24-hydroxyl-epothilone B, based on MS and NMR data (compared with
data of epothilone B): 5
[0136] Molecular Formula: C.sub.27H.sub.41NO.sub.7S
[0137] Molecular Weight: 523
[0138] Mass Spectrum: ES+(m/z): 524([M+H].sup.+), 506.
[0139] LC/MS/MS: +ESI (m/z): 524, 506, 476, 436, 320
[0140] HRMS: Calculated for [M+H].sup.+: 524.2682; Found:
524.2701.
[0141] HPLC (Rt) 7.3 minutes (on the analytical HPLC system)
[0142] LC/NMR Observed Chemical Shifts Varian AS-600 (Proton:
599.624 MHz), Solvent D.sub.2O/CD.sub.3CN (.delta.1.94): .about.4/6
Proton: .delta.7.30 (s, 1H), 6.43 (s, 1H), 5.30 (m, 1H), 4.35 (m,
1H), 3.81 (m, 1H), 3.74 (m, 1H), 3.68 (m, 1H), 3.43 (m, 1H), 2.87
(m, 1H), 2.66 (s, 3H), 2.40 (m, 2H), 1.58 (b, 1H), 1.48 (b, 1H),
1.35 (m, 3H), 1.18 (s, 3H), 1.13 (s, 3H), 0.87 (m, 6H) *Peaks
between 1.8-2.1 ppm were not observed due to solvent
suppression.
[0143] The proton chemical shift was assigned as follows:
7 Position Proton Pattern 1 -- 2 2.40 m 3 4.35 m 4 -- 5 -- 6 3.43 m
7 3.68 m 8 1.58 m 9 1.35 b 10 1.48 b 10 1.35 b 11 SSP 12 -- 13 2.87
m 14 SSP 15 5.30 m 16 -- 17 6.43 s 18 -- 19 7.30 s 20 -- 21 2.66 s
22 1.18 s 23 0.87 m 24 3.81 m 24 3.74 m 25 0.87 m 26 1.13 s 27 SSP
*SSP: no observed due to solvent suppression.
[0144] Accordingly, the compositions and methods of the present
invention are useful in producing known compounds that are
microtubule-stabilizing agents as well as new compounds comprising
epothilone analogs such as 24-hydroxyl-epothilone B (Formula A) and
pharmaceutically acceptable salts thereof expected to be useful as
microtubule-stabilizing agents. The microtubule stabilizing agents
produced using these compositions and methods are useful in the
treatment of a variety of cancers and other proliferative diseases
including, but not limited to, the following;
[0145] carcinoma, including that of the bladder, breast, colon,
kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and
skin; including squamous cell carcinoma;
[0146] hematopoietic tumors of lymphoid lineage, including
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins
lymphoma, hairy cell lymphoma and Burketts lymphoma;
[0147] hematopoietic tumors of myeloid lineage, including acute and
chronic myelogenous leukemias and promyelocytic leukemia;
[0148] tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyoscarcoma;
[0149] other tumors, including melanoma, seminoma,
tetratocarcinoma, neuroblastoma and glioma;
[0150] tumors of the central and peripheral nervous system,
including astrocytoma, neuroblastoma, glioma, and schwannomas;
[0151] tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyosarcoma, and osteosarcoma; and
[0152] other tumors, including melanoma, xenoderma pigmentosum,
keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma.
[0153] Microtubule stabilizing agents produced using the
compositions and methods of the present invention will also inhibit
angiogenesis, thereby affecting the growth of tumors and providing
treatment of tumors and tumor-related disorders. Such
anti-angiogenesis properties of these compounds will also be useful
in the treatment of other conditions responsive to
anti-angiogenesis agents including, but not limited to, certain
forms of blindness related to retinal vascularization, arthritis,
especially inflammatory arthritis, multiple sclerosis, restinosis
and psoriasis.
[0154] Microtubule stabilizing agents produced using the
compositions and methods of the present invention will induce or
inhibit apoptosis, a physiological cell death process critical for
normal development and homeostasis. Alterations of apoptotic
pathways contribute to the pathogenesis of a variety of human
diseases. Compounds of the present invention such as those set
forth in formula I and II and Formula A, as modulators of
apoptosis, will be useful in the treatment of a variety of human
diseases with aberrations in apoptosis including, but not limited
to, cancer and precancerous lesions, immune response related
diseases, viral infections, degenerative diseases of the
musculoskeletal system and kidney disease.
[0155] Without wishing to be bound to any mechanism or morphology,
microtubule stabilizing agents produced using the compositions and
methods of the present invention may also be used to treat
conditions other than cancer or other proliferative diseases. Such
conditions include, but are not limited to viral infections such as
herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and
adenovirus; autoimmune diseases such as systemic lupus
erythematosus, immune mediated glomerulonephritis, rheumatoid
arthritis, psoriasis, inflammatory bowel diseases and autoimmune
diabetes mellitus; neurodegenerative disorders such as Alzheimer's
disease, AIDS-related dementia, Parkinson's disease, amyotrophic
lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy
and cerebellar degeneration; AIDS; myelodysplastic syndromes;
aplastic anemia; ischemic injury associated myocardial infarctions;
stroke and reperfusion injury; restenosis; arrhythmia;
atherosclerosis; toxin-induced or alcohol induced liver diseases;
hematological diseases such as chronic anemia and aplastic anemia;
degenerative diseases of the musculoskeletal system such as
osteoporosis and arthritis; aspirin-sensitive rhinosinusitis;
cystic fibrosis; multiple sclerosis; kidney diseases; and cancer
pain.
[0156] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
[0157] Reagents
[0158] R2 Medium was Prepared as Follows:
[0159] A solution containing sucrose (103 grams), K.sub.2SO.sub.4
(0.25 grams) MgCl.sub.2.6H.sub.2O (10.12 grams), glucose (10
grams), Difco Casaminoacids (0.1 grams) and distilled water (800
ml) was prepared. Eighty ml of this solution was then poured into a
200 ml screw capped bottle containing 2.2 grams Difco Bacto agar.
The bottle was capped and autoclaved. At time of use, the medium
was remelted and the following autoclaved solutions were added in
the order listed:
[0160] 1 ml KH.sub.2PO.sub.4 (0.5%)
[0161] 8 ml CaCl.sub.2.2H.sub.2O (3.68%)
[0162] 1.5 ml L-proline (20%)
[0163] 10 ml TES buffer (5.73%, adjusted to pH 7.2)
[0164] 0.2 ml Trace element solution containing ZnCl.sub.2(40 mg),
FeCl.sub.3.6H.sub.2O(200 mg), CuCl.sub.2.2H.sub.2O (10 mg),
MnCl.sub.2.4H.sub.2O (10 mg), Na.sub.2B.sub.4O.sub.7.10H.sub.2O (10
mg), and (NH.sub.4).sub.6Mo.sub.7O.sub.24.H.sub.2O
[0165] 0.5 ml NaOH (1N)(sterilization not required)
[0166] 0.5 ml Required growth factors for auxotrophs (Histidine (50
.mu.g/ml); Cysteine (37 .mu.g/ml); adenine, guanine, thymidine and
uracil (7.5 .mu.g/ml); and Vitamins (0.5 .mu.g/ml).
[0167] R2YE medium was prepared in the same fashion as R2 medium.
However, 5 ml of Difco yeast extract (10%) was added to each 100 ml
flask at time of use.
[0168] P (protoplast) buffer was prepared as follows:
[0169] A basal solution made up of the following was prepared:
[0170] Sucrose (103 grams)
[0171] K.sub.2SO.sub.4 (0.25 grams)
[0172] MgCl.sub.2.6H.sub.2O (2.02 grams)
[0173] MgCl.sub.2.6H.sub.2O (2.02 grams)
[0174] Trace Element Solution as described for R2 medium (2 ml)
[0175] Distilled water to 800 ml
[0176] Eighty ml aliquots of the basal solution were then dispensed
and autoclaved. Before use, the following was added to each flask
in the order listed:
[0177] 1 ml KH.sub.2PO.sub.4 (0.5%)
[0178] 10 ml CaCl.sub.2.2H.sub.2O (3.68%)
[0179] TES buffer (5.75%, adjusted to pH 7.2)
[0180] T (transformation) buffer was prepared by mixing the
following sterile solutions:
[0181] 25 ml Sucrose (10.3%)
[0182] 75 ml distilled water
[0183] 1 ml Trace Element Solution as described for R2 medium
[0184] 1 ml K.sub.2SO.sub.4 (2.5%)
[0185] The following are then added to 9.3 mls of this
solution:
[0186] 0.2 ml CaCl.sub.2 (5M)
[0187] 0.5 ml Tris maleic acid buffer prepared from 1 M solution of
Tris adjusted to pH 8.0 by adding maleic acid.
[0188] For use, 3 parts by volume of the above solution are added
to 1 part by weight of PEG 1000, previously sterilized by
autoclaving.
[0189] L (lysis) buffer was prepared by mixing the following
sterile solutions:
[0190] 100 ml Sucrose (10.3%)
[0191] 10 ml TES buffer (5.73%, adjusted to pH 7.2)
[0192] 1 ml K.sub.2SO.sub.4 (2.5%)
[0193] 1 ml Trace Element Solution as described for R2 medium
[0194] 1 ml KH.sub.2PO.sub.4 (0.5%)
[0195] 0.1 ml MgCl.sub.2.6H.sub.2O (2.5 M)
[0196] 1 ml CaCl.sub.2 (0.25 M)
[0197] CRM Medium
[0198] A solution containing the following components was prepared
in 1 liter of dH.sub.2O: glucose (10 grams), sucrose (103 grams),
MgCl.sub.2.6H.sub.2O (10.12 grams), BBL.TM. trypticase soy broth
(15 grams) (Becton Dickinson Microbiology Systems, Sparks, Md.,
USA), and BBL.TM. yeast extract (5 grams) (Becton Dickinson
Microbiology Systems). The solution was autoclaved for 30 minutes.
Thiostrepton was added to a concentration of 10 .mu.g/ml for
cultures propagated with plasmids.
[0199] Electroporation Buffer
[0200] A solution containing 30% (wt/vol) PEG 1000, 10% glycerol,
and 6.5% sucrose was prepared in dH.sub.2O. The solution was
sterilized by vacuum filtration through a 0.22 .mu.m cellulose
acetate filter.
Example 2
[0201] Extraction of Chromosomal DNA from Strain SC15847
[0202] Genomic DNA was isolated from an Amycolatopsis orientalis
soil isolate strain designation SC15847 (ATCC PT-1043) using a
guanidine-detergent lysis method, DNAzol reagent (Invitrogen,
Carlsbad, Calif., USA). The SC15847 culture was grown 24 hours at
28.degree. C. in F7 medium (glucose 2.2%, yeast extract 1.0%, malt
extract 1.0%, peptone 0.1%, pH 7.0). Twenty ml of culture was
harvested by centrifugation and resuspended in 20 ml of DNAzol,
mixed by pipetting and centrifuged 10 minutes in the Beckman TJ6
centrifuge. Ten ml of 100% ethanol was added, inverted several
times and stored at room temperature 3 minutes. The DNA was spooled
on a glass pipette washed in 100% ethanol and allowed to air dry 10
minutes. The pellet was resuspended in 500 .mu.l of 8 mM NaOH and
once dissolved it was neutralized with 30 .mu.l of 1M HEPES
pH7.2.
Example 3
[0203] PCR Reactions
[0204] PCR reactions were prepared in a volume of 50 .mu.l,
containing 200-500 ng of genomic DNA or 1.0 .mu.l of the cDNA, a
forward and reverse primer, and the forward primer being either
P450-1.sup.+ (SEQ ID NO:23) or P450-1a.sup.+ (SEQ ID NO:24) or
P450-2.sup.+ (SEQ ID NO:25) and the reverse primer P450-3.sup.-
(SEQ ID NO:27) or P450-2.sup.- (SEQ ID NO:26). All primers were
added to a final concentration of 1.4-2.0 .mu.M. The PCR reaction
was prepared with 1 .mu.l of Taq enzyme (2.5 units) (Stratagene), 5
.mu.l of Taq buffer and 4 .mu.l of 2.5 mM of dNTPs with dH.sub.2O
to 50 .mu.l. The cycling reactions were performed on a Geneamp.RTM.
PCR system with the following protocol: 95.degree. C. for 5
minutes, 5 cycles [95.degree. C. 30 seconds, 37.degree. C. 15
seconds (30% ramp), 72.degree. C. 30 seconds], 35 cycles
(94.degree. C. 30 seconds, 65.degree. C. 15 seconds, 72.degree. C.
30 seconds), 72.degree. C. 7 minutes. The expected sizes for the
reactions are 340 bp for the P450-1.sup.+ (SEQ ID NO:23) or
P450-1a.sup.+ (SEQ ID NO:24) and P450-3.sup.- (SEQ ID NO:27) primer
pairs, 240 bp for the P450-1.sup.+ (SEQ ID NO:23) and
P450-2.sup.-(SEQID NO:26) primer pairs and 130 bp for the
P450-2.sup.+ (SEQ ID NO:25) and P450-3.sup.- (SEQ ID NO:27) primer
pairs.
Example 4
[0205] Cloning of Epothilone B Hydroxylase and Ferredoxin Genes
[0206] Twenty .mu.g of SC15847 genomic DNA was digested with BglII
restriction enzyme for 6 hours at 37.degree. C. A 30 k nanosep
column (Gelman Sciences, Ann Arbor, Mich., USA) was used to
concentrate the DNA and remove the enzyme and buffer. The reactions
were concentrated to 40 .mu.l and washed with 200 .mu.l of TE. The
digestion products were then separated a 0.7% agarose gel and
genomic DNA in the range of 12.about.15 kb was excised from the gel
and purified using the Qiagen gel extraction method. The genomic
DNA was then ligated to plasmid pWB19N (U.S. Pat. No. 5,516,679),
which had been digested with BamHI and dephosphorylated using the
SAP I enzyme (Roche Molecular Biochemicals, Indianapolis, Ind.,
catalog#1 758 250). Ligation reactions were performed in a 15 .mu.l
volume with 1 U of T4 DNA ligase (Invitrogen) for 1 hour at room
temperature. One .mu.l of the ligation was transformed to 100 .mu.l
of chemically competent DH10B cells (Invitrogen) and 100 .mu.l
plated to five LB agar plates with 30 .mu.g/ml of neomycin,
37.degree. C. overnight.
[0207] Five nylon membrane circles (Roche Molecular Biochemicals,
Indianapolis, Ind.) were numbered and marked for orientation. The
membranes were placed on the plates 2 minutes and then allowed to
dry for 5 minutes. The membranes were then placed on Whatman filter
disks saturated with 10% SDS for 5 minutes, 0.5N NaOH with 1.5 M
NaCl for 5 minutes, 1.5 M NaCl with 1.0 M Tris pH 8.0 for 5
minutes, and 15 minutes on 2.times.SSC. The filters were hybridized
as described previously for the Southern hybridization. Hybridizing
colonies were picked to 2 ml of TB with 30 .mu.g/ml neomycin and
grown overnight at 37.degree. C. Plasmid DNA was isolated using a
miniprep column procedure (Mo Bio). This plasmid was named
NPB29-1.
Example 5
[0208] DNA Sequencing and Analysis
[0209] The cloned PCR products were sequenced using
fluorescent-dye-labeled terminator cycle sequencing, Big-Dye
sequencing kit (Applied Biosystems, Foster city, Calif., USA) and
were analyzed using laser-induced fluorescence capillary
electrophoresis, ABI Prism 310 sequencer (Applied Biosystems).
Example 6
[0210] Extraction of Total RNA
[0211] Total RNA was isolated from the SC15847 culture using a
modification of the Chomczynski and Sacchi method with a
mono-phasic solution of phenol and guanidine isothiocyanate, Trizol
reagent (Invitrogen). Five ml of an SC 15847 frozen stock culture
was thawed and used to inoculate 100 ml of F7 media in a 500 ml
Erlenmeyer flask. The culture was grown in a shaker incubator at
230 rpm, 30.degree. C. for 20 hours to an optical density at 600 nm
(OD.sub.600) of 9.0. The culture was placed in a 16.degree. C.
shaker incubator at 230 rpm for 20 minutes. Fifty-five milligrams
of epothilone B was dissolved in 1 ml of 100% ethanol and added to
the culture. A second ml of ethanol was used to rinse the residual
epothilone B from the tube and added to the culture. The culture
was incubated at 16.degree. C., 230 rpm for 30 hours. Thirty ml of
the culture was transferred to a 50 ml tube, 150 mg of lysozyme was
added to the culture and the culture was incubated 5 minutes at
room temperature. Ten ml of the culture was placed in a 50 ml
Falcon tube and centrifuged 5 minutes, 4.degree. C. in a TJ6
centrifuge. Two ml of chloroform was added and the tube was mixed
vigorously for 15 seconds. The tube was incubated 2 minutes at room
temperature and centrifuged 10 minutes, top speed in the TJ6
centrifuge. The aqueous layer was transferred to a fresh tube and
2.5 ml of isopropanol was added to precipitate the RNA. The tube
was incubated 10 minutes at room temperature and centrifuged 10
minutes, 4.degree. C. The supernatant was removed, the pellet was
rinsed with 70% ethanol and dried briefly under vacuum. The pellet
was resuspended in 150 .mu.l of RNase-free dH.sub.2O. Fifty .mu.l
of 7.5M LiCl was added to the RNA and incubated at -20.degree. C.
for 30 minutes. The RNA was pelleted by centrifugation 10 minutes,
4.degree. C. in a microcentrifuge. The pellet was rinsed with 200
.mu.l of 70% ethanol, dried briefly under vacuum and resuspended in
150 .mu.l of RNase free dH.sub.2O.
[0212] The RNA was treated with DNaseI (Ambion, Austin, Tex., USA).
Twenty-five .mu.l of total RNA (5.3 .mu.g/.mu.l), 2.5 .mu.l of
DNaseI buffer, 1.0 .mu.l of DNase I added and incubated at
37.degree. C. for 25 minutes. Five .mu.l of DNase I inactivation
buffer added, incubated 2 minutes, centrifuged 1 minute, the
supernatant was transferred to a fresh tube.
Example 7
[0213] cDNA Synthesis
[0214] cDNA was synthesized from the total RNA using the
Superscript II enzyme (Invitrogen). The reaction was prepared with
1 .mu.l of total RNA (5.3 .mu.g/.mu.l), 9 .mu.l of dH.sub.2O, 1
.mu.l of dNTP mix (10 mM), and 1 .mu.l of random hexamers. The
reaction was incubated at 65.degree. C. for 5 minutes then placed
on ice. The following components were then added: 4 .mu.l of
1.sup.st strand buffer, 1 .mu.l of RNase Inhibitor, 2.0 .mu.l of
0.1 M DTT, and 1 .mu.l of Superscript II enzyme. The reaction was
incubated at room temperature 10 minutes, 42.degree. C. for 50
minutes and 70.degree. C. for 15 minutes. One .mu.l of RNaseH was
added and incubated 20 minutes at 37.degree. C., 15 minutes at
70.degree. C. and stored at 4.degree. C.
Example 8
[0215] DNA Labeling
[0216] The PCR conditions used to amplify the P450 specific
products from genomic DNA and cDNA were used to amplify the insert
of plasmid pCRscript-29. Plasmid pCRscript-29 contains a 340 bp PCR
fragment amplified from SC15847 genomic DNA using primers P450
1.sup.+ (SEQ ID NO:23) and P450 3.sup.- (SEQ ID NO:27). Two .mu.l
of the plasmid prep was used as a template, with a total of 25
cycles. The amplified product was gel purified using the Qiaquick
gel extraction system (Qiagen). The extracted DNA was ethanol
precipitated and resuspended in 5 .mu.l of TE, the yield was
estimated to be 500 ng. This fragment was labeled with digoxigenin
using the chem link labeling reagent (Roche Molecular Biochemicals,
Indianapolis, Ind. catalog #1 836 463). Five .mu.l of the PCR
product was mixed with 0.5 .mu.l of Dig-chem link and dH.sub.2O
added to 20 .mu.l. The reaction was incubated 30 minutes at
85.degree. C. and 5 .mu.l of stop solution added. The probe
concentration was estimated at 20 ng/.mu.l.
Example 9
[0217] Southern DNA Hybridization
[0218] Ten .mu.l of genomic DNA (0.5 .mu.g/.mu.l) was digested with
BamHI, BglII, EcoRI, HindIII or NotI and separated at 12 volts for
16 hours. The gel was depurinated 10 minutes in 0.25 N HCl and
transferred by vacuum to a nylon membrane (Roche Molecular
Biochemicals) in 0.4 N NaOH 5" Hg, 90 minutes using a vacuum
blotter (Bio-Rad Laboratories, Inc. Hercules, Calif., USA catalog
#165-5000). The membrane was rinsed in 1 M ammonium acetate and
UV-crosslinked using the Stratalinker UV Crosslinker (Stratagene).
The membrane was rinsed in 2.times.SSC and stored at room
temperature.
[0219] The membrane was prehybridized 1 hour at 42.degree. C. in 20
ml of Dig Easy Hyb buffer (Roche Molecular Biochemicals). The probe
was denatured 10 minutes at 65.degree. C. and then placed on ice.
Five ml of probe in Dig-Easy Hyb at an approximate concentration on
20 ng/ml was incubated with the membrane at 42.degree. C.
overnight. The membrane was washed 2 times in 2.times.SCC with 0.1%
SDS at room temperature, then 2 times in 0.5.times.SSC with 0.1%
SDS at 65.degree. C. The membrane was equilibrated in Genius buffer
1 (10 mM maleic acid, 15 mM NaCl; pH 7.5; 0.3% v/v Tween 20) (Roche
Molecular Biochemicals, Indianapolis, Ind.) for 2 minutes, then
incubated with 2% blocking solution (2% Blocking reagent in Genius
Buffer 1 )(Roche Molecular Biochemicals Indianapolis, Ind.) for 1
hour at room temperature. The membrane was incubated with a
1:20,000 dilution of anti-dig antibody in 50 ml of blocking
solution for 30 minutes. The membrane was washed 2 times, 15
minutes each in 50 ml of Genius buffer 1. The membrane was
equilibrated for two minutes in Genius Buffer 3 (10 mM Tris-HCl,
10mM NaCl; pH 9.5). One ml of a 1:100 dilution of CSPD (disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricycl-
o[3.3.1.1.sup.3.7]decan}-4-yl)phenyl phosphate) (Roche Molecular
Biochemicals) in Genius buffer 3 was added to the membrane and
incubated 5 minutes at room temperature, then placed at 37.degree.
C. for 15 minutes. The membrane was exposed to Biomax ML film
(Kodak, Rochester, N.Y., USA) for 1 hour.
Example 10
[0220] E. coli Transformation
[0221] Competent cells were purchased from Invitrogen. E. coli
strain DH10B was used as a host for genomic cloning. The chemically
competent cells were thawed on ice and 100 .mu.l aliquoted to a
17.times.100-mm polypropylene tube on ice. One .mu.l of the
ligation mixture was added to the cells and incubated on ice for 30
minutes. The cells were incubated at 42.degree. C. for 45 seconds,
then placed on ice 1-2 minutes. 0.9 ml pf SOC. medium(Invitrogen)
was added and the cells were incubated one hour at 30-37.degree. C.
at 200-240 rpm. Cells were plated on a selective medium (Luria agar
with neomycin or ampicillin at a concentration of 30 .mu.g/ml or
100 .mu.g/ml respectively).
Example 11
[0222] Transformation of Streptomyces lividans TK24
[0223] Plasmid pWB19N849 was constructed by digesting plasmid
pWB19N with HindIII and treating with SAP I and digesting plasmid
pANT849 (Keiser, et al., 2000, Practical Streptomyces Genetics,
John Innes ) with HindIII. The two linearized fragments were
ligated 1 hour at room temperature with 1 U of T4 DNA ligase. One
.mu.l of the ligation reaction was used to transform XL-1 Blue
electrocompetent cells (Stratagene). The recovered cells were
plated to LB neomycin (30 .mu.g/ml) overnight at 37.degree. C.
Colonies were picked to 2 ml of LB with 30 .mu.g/ml neomycin and
incubated overnight at 30.degree. C. MoBio plasmid minipreps were
performed on all cultures. Plasmids constructed from the ligation
of pWB19N and pANT849 were determined by electrophoretic mobility
on 0.7% agarose. The plasmid pWB19N849 was digested with HindIII
and BglII to excise a 5.3 kb fragment equivalent to plasmid pANT849
digested with BglII and HindIII. This 5.3 kb fragment was purified
on an agarose gel and extracted using the Qiaquick gel extraction
system.
[0224] A 1.469 kb DNA fragment containing the epothilone B
hydroxylase gene and the downstream ferredoxin gene was amplified
using PCR. The 50 .mu.l PCR reaction was composed of 5 .mu.l of Taq
buffer, 2.5 .mu.l glycerol, 1 .mu.l of 20 ng/.mu.l NPB29-1 plasmid,
0.4 .mu.l of 25 mM dNTPs, 1.0 .mu.l each of primers NPB29-6F (SEQ
ID NO:28) and NPB29-7R (SEQ ID NO:29) (5 pmole/.mu.l), 38.1 .mu.l
of dH.sub.2O and 0.5 .mu.l of Taq enzyme (Stratagene). The
reactions were performed on a Perkin Elmer 9700, 95.degree. C. for
5 minutes, then 30 cycles (96.degree. C. for 30 seconds, 60.degree.
C. 30 seconds, 72.degree. C. for 2 minutes), and 72.degree. C. for
7 minutes. The PCR product was purified using a Qiagen minielute
column with the PCR cleanup procedure. The purified product was
digested with BglII and HindIII and purified on a 0.7% agarose gel.
A 1.469 kb band was excised from the gel and eluted using a Qiagen
minielute column. Five .mu.l of this PCR product was ligated with 2
.mu.l of the BglII, HindIII digested pANT849 vector in a 10 .mu.l
ligation reaction. The reaction was incubated at room temperature
for 24 hours and then transformed to S. lividans TK24
protoplasts.
[0225] Twenty ml of YEME media was inoculated with a frozen spore
suspension of S. lividans TK24 and grown 48 hours in a 125 ml
bi-indent flask. Protoplasts were prepared as described in
Practical Streptomyces Genetics. The ligation reaction was mixed
with protoplasts, then 500 .mu.l of transformation buffer was
added, followed immediately by 5 ml of P buffer. The transformation
reactions were spun down 7 minutes at 2,750 rpm, resuspended in 100
.mu.l of P buffer and plated to one R2YE plate. The plate was
incubated at 28.degree. C. for 20 hours then overlaid with 5 ml of
LB 0.7% agar with 250 .mu.g/ml thiostrepton. After 7 days colonies
were picked to an R2YE grid plate with 50 .mu.g/ml of thiostrepton.
The colonies were grown an additional 5 days at 28.degree. C., then
stored at 4.degree. C.
[0226] This recombinant microorganism has been deposited with the
ATCC and designated PTA-4022.
Example 12
[0227] Transformation of Streptomyces rimosus
[0228] The procedure of Pigac and Schrempf Appl. Environ Microb.,
Vol. 61, No. 1, 352-356 (1995) was used to transform S. rimosus. S.
rimosus strain R6 593 was cultivated in 20 ml of CRM medium at
30.degree. C. on a rotary shaker (250 rpm). The cells were
harvested at 24 hrs by centrifugation for 5 minutes, 5,000 rpm,
4.degree. C., and resuspended in 20 ml of 10% sucrose, 4.degree.
C., and centrifuged for 5 minutes, 5,000 rpm, 4.degree. C. The
pellet was resuspended in 10 ml of 15% glycerol, 4.degree. C. and
centrifuged for 5 minutes, 5,000 rpm, 4.degree. C. The pellet was
resuspended in 2 ml of 15% glycerol, 4.degree. C. with 100 .mu.g/ml
lysozyme and incubated at 37.degree. C. for 30 minutes, centrifuged
for 5 minutes, 5,000 rpm, 4.degree. C. and resuspended in 2 ml of
15% glycerol, 4.degree. C. The 15% glycerol wash was repeated once
and the pellet was resuspended in 1 to 2 ml of Electroporation
Buffer. The cells were stored at -80.degree. C. in 50-200 .mu.l
aliquots.
[0229] The ligations were prepared as described for the S. lividans
transformation. After the incubation of the ligation reaction, the
volume was brought to 100 .mu.l with dH.sub.2O, NaCl was added to
0.3M, and the reaction extracted with an equal volume of 24:1:1
phenol:choroform isoamyl alcohol. Twenty .mu.g of glycogen was
added and the ligated DNA was precipitated with 2 volumes of 100%
ethanol at -20.degree. C. for 30 minutes. The DNA was pelleted 10
minutes in a microcentrifuge, washed once with 70% ethanol, dried 5
minutes in a speed-vac concentrator and resuspended in 5 .mu.l of
dH.sub.2O.
[0230] One frozen aliquot of cells was thawed at room temperature
and divided, 50 .mu.l tube for each DNA sample for electroporation.
The cells were stored on ice until use. DNA in 1 to 2 .mu.l of
dH.sub.2O was added and mixed. The cell and DNA mixture was
transferred to a 2 mm gapped electrocuvette (Bio-Rad Laboratories,
Richmond Calif. USA) that was pre-chilled on ice. The cells were
electroporated at a setting of 2 kV (10 kV/cm), 25 .mu.F,
400.OMEGA. using a Gene Pulser.TM. (Bio-Rad Laboratories). The
cells were diluted with 0.75 to 1.0 ml of CRM (0-4.degree. C.),
transferred to 15 ml culture tubes and incubated with agitation 3
hrs at 30.degree. C. The cells were plated on trypticase soy broth
agar plates with 10-30 .mu.g/ml of thiostrepton.
Example 13:
[0231] High Performance Liquid Chromatography
[0232] The liquid chromatography separation was performed using a
Waters 2690 Separation Module system (Waters Corp., Milford, Mass.,
USA) and a column, 4.6.times.150 mm, filled with SymmetryShield
RP.sub.8, particle size 3.5 .mu.m (Waters Corp., Milford, Mass.,
USA). The gradient mobile phase programming was used with a flow
rate of 1.0 ml/minute. Eluent A was water/acetonitrile (20:1)+10 mM
ammonium acetate. Eluent B was acetonitrile/water (20:1). The
mobile phase was a linear gradient from 12% B to 28% B over 6
minutes and held isocratic at 28% B over 4 minutes. This was
followed by a 28% B to 100% B linear gradient over 20 minutes and a
linear gradient to 12% B over two minutes with a 3 minute hold at
12% B.
Example 14:
[0233] Mass Spectrometry
[0234] The column effluent was introduced directly into the
electrospray ion source of a ZMD mass spectrometer (Micromass,
Manchester, UK). The instrument was calibrated using Test Juice
reference standard (Waters Corp, Milford, Mass., USA) and was
delivered at a flow of 10 .mu.l/minute from a syringe pump (Harvard
Apparatus, Holliston, Mass., USA). The mass spectrometer was
operated at a low mass resolution of 13.2 and a high mass
resolution of 11.2. Spectra were acquired from using a scan range
of m/z 100 to 600 at an acquisition rate of 10 spectra/second. The
ionization technique employed was positive electrospray (ES). The
sprayer voltage was kept at 2900 V and the cone voltage of the ion
source was kept at a potential of 17 V.
Example 15
[0235] Use of the ebh Gene Sequence (SEQ ID NO:1) to Isolate
Cytochrome P450 Genes from Other Microorganisms
[0236] Genomic DNA was isolated from a set of cultures (ATCC43491,
ATCC14930, ATCC53630, ATCC53550, ATCC39444, ATCC43333, ATCC35165)
using the DNAzol reagent. The DNA was used as a template for PCR
reactions using primers designed to the sequence of the ebh gene.
Three sets of primers were used for amplification; NPB29-6f (SEQ ID
NO:28) and NPB29-7r (SEQ ID NO:29), NPB29-16f (SEQ ID NO:50) and
NPB29-17r (SEQ ID NO:51), and NPB29-19f (SEQ ID NO:52) and
NPB29-20r (SEQ ID NO:53).
[0237] PCR reactions were prepared in a volume of 20 .mu.l,
containing 200-500 ng of genomic DNA and a forward and reverse
primer. All primers were added to a final concentration of 1.4-2.0
.mu.M. The PCR reaction was prepared with 0.2 .mu.l of
Advantage.TM. 2 Taq enzyme (BD Biosciences Clontech, Palo Alto,
Calif., USA), 2 .mu.l of Advantage.TM. 2 Taq buffer and 0.2 .mu.l
of 2.5 mM of dNTPs with dH.sub.2O to 20 .mu.l. The cycling
reactions were performed on a Geneamp.RTM. 9700 PCR system or a
Mastercycler.RTM. gradient (Eppendorf, Westbury, N.Y., USA) with
the following protocol: 95.degree. C. for 5 minutes, 35 cycles
(96.degree. C. 20 seconds, 54-69.degree. C. 30 seconds, 72.degree.
C. 2 minutes), 72.degree. C. for 7 minutes. The expected size of
the PCR products is approximately 1469 bp for the NPB29-6f (SEQ ID
NO:28) and NPB29-7r (SEQ ID NO:29) primer pair, 1034 bp for the
NPB29-16f (SEQ ID NO:50) and NPB29-17r (SEQ ID NO:51) primer pair
and 1318 bp for the NPB29-19f (SEQ ID NO:52) and NPB29-20r (SEQ ID
NO:53) primer pair. The PCR reactions were analyzed on 0.7% agarose
gels. PCR products of the expected size were excised from the gel
and purified using the Qiagen gel extraction method. The purified
products were sequenced using the Big-Dye sequencing kit and
analyzed using an ABI310 sequencer.
Example 16
[0238] Construction of Plasmid pPCRscript-ebh
[0239] A 1.469 kb DNA fragment containing the epothilone B
hydroxylase gene and the downstream ferredoxin gene was amplified
using PCR. The 50 .mu.l PCR reaction was composed of 5 .mu.l of Taq
buffer, 2.5 .mu.l glycerol, 1 .mu.l of 20 ng/.mu.l NPB29-1 plasmid,
0.4 .mu.l of 25 mM dNTPs, 1.0 .mu.l each of primers NPB29-6f (SEQ
ID NO:28) and NPB29-7r (SEQ ID NO:29) (5 pmole/.mu.l), 38.1 .mu.l
of dH.sub.2O and 0.5 .mu.l of Taq enzyme (Stratagene). The
reactions were performed on a Geneampe.RTM. 9700 PCR system, with
the following conditions; 95.degree. C. for 5 minutes, then 30
cycles (96.degree. C. for 30 seconds, 60.degree. C. 30 seconds,
72.degree. C. for 2 minutes), and 72.degree. C. for 7 minutes. The
PC product was purified using a Qiagen Qiaquick column with the PCR
cleanup procedure. The purified product was digested with BglII and
HindIII and purified on a 0.7% agarose gel. A 1.469 kb band was
excised from the gel and eluted using a Qiagen Qiaquick gel
extraction procedure. The fragments were then cloned into the
pPCRscript Amp vector using the PCRscript Amp cloning kit. Colonies
containing inserts were picked to 1-2 ml of LB (Luria Broth) with
100 .mu.g/ml ampicillin, 30-37.degree. C., 16-24 hours, 230-300
rpm. Plasmid isolation was performed using the Mo Bio miniplasmid
prep kit. The sequence of the insert was confirmed by cycle
sequencing with the Big-Dye sequencing kit. This plasmid was named
pPCRscript-ebh.
Example 17
[0240] Mutagenesis of the ebh Gene for Improved Yield or Altered
Specificity
[0241] The Quikchange.RTM. XL Site-Directed Mutagenesis Kit and the
Quikchange.RTM. Multi Site-Directed Mutagenesis kit, both from
Stratagene were used to introduce mutations in the coding region of
the ebh gene. Both of these methods employ DNA primers 35-45, bases
in length containing the desired mutation (SEQ ID NO:54-59 and 77),
a methylated circular plasmid template and PfuTurbo.RTM. DNA
Polymerase (U.S. Pat. Nos. 5,545,552 and 5,866,395 and 5,948,663)
to generate copies of the plasmid template incorporating the
mutation carried on the mutagenic primers. Subsequent digestion of
the reaction with the restriction endonuclease enzyme DpnI,
selectively digests the methylated plasmid template, but leaves the
non-methylated mutated plasmid intact. The manufacturer's
instructions were followed for all procedures with the exception of
the DpnI digestion step in which the incubation time was increased
from 1 hr to 3 hrs. The pPCRscript-ebh vector was used as the
template for mutagenesis.
[0242] One to two .mu.l of the reaction was transformed to either
XL1-Blue.RTM. electrocompetent or XL10-Gold.RTM. ultracompetent
cells (Stratagene). Cells were plated to a density of greater than
100 colonies per plate on LA (Luria Agar) 100 .mu.g/ml ampicillin
plates, and incubated 24-48 hrs at 30-37.degree. C. The entire
plate was resuspended in 5 ml of LB containing 100 .mu.g/ml
ampicillin. Plasmid was isolated directly from the resuspended
cells by centrifuging the cells and then purifying the plasmid
using the Mo Bio miniprep procedure. This plasmid was then used as
a template for PCR with primers NPB29-6f (SEQ ID NO:28)and NPB29-7r
(SEQ ID NO:29) to amplify a mutated expression cassette. Digestion
of the 1.469 kb PCR product with the restriction enzymes BglII and
HindIII was used to prepare this fragment for ligation to vector
pANT849 also digested with BglII and HindIII. Alternatively, the
resuspended cells were used to inoculate 20-50 ml of LB containing
100 .mu.g/ml ampicillin and grown 18-24 hrs at 30-37.degree. C.
Qiagen midi-preps were performed on the cultures to isolate plasmid
DNA containing the desired mutation. Digestion with the restriction
enzymes BglII and HindIII was used to excise the mutated expression
cassette for ligation to BglII and HindIII digested plasmid
pANT849. Screening of mutants was performed in S. lividans or S.
rimosus as described.
[0243] Alternatively, the method of Leung et al., Technique--A
Journal of Methods in Cell and Molecular Biology, Vol. 1, No. 1,
11-15 (1989) was used to generate random mutation libraries of the
ebh gene. Manganese and/or reduced dATP concentration is used to
control the mutagenesis frequency of the Taq polymerase. The
plasmid pCRscript-ebh was digested with NotI to linearize the
plasmid. The Polymerase buffer was prepared with 0.166 M
(NH.sub.4).sub.2SO.sub.4, 0.67M Tris-HCl pH 8.8, 61 mM MgCl.sub.2,
67 .mu.M EDTA pH8.0, 1.7 mg/ml Bovine Serum Albumin). The PCR
reaction was prepared with 10 .mu.l of Not I digested pCRscript-ebh
(0.1 ng/.mu.l), 10 .mu.l of polymerase buffer, 1.0 .mu.l of 1M
.beta.-mercaptoethanol, 10.0 .mu.l of DMSO, 1.0 .mu.l of NPB29-6f
(SEQ ID NO:28) primer (100 pmole/.mu.l), 1.0 .mu.l of NPB29-7r (SEQ
ID NO:29) primer (100 pmole/.mu.l), 10 .mu.l of 5 mM MnCl.sub.2,
10.0 .mu.l 10 mM dGTP, 10.0 .mu.l 2 mM dATP, 10 mM dTTP, 10.0 .mu.l
10 mM dCTP, and 2.0 .mu.l Taq polymerase. dH.sub.2O was added to
100 .mu.l. Reactions were also prepared as described above but
without MnCl.sub.2. The cycling reactions were performed a
GeneAmp.RTM. PCR system with the following protocol: 95.degree. C.
for 1 minute, 25-30 cycles(94.degree. C. for 1 minute, 55.degree.
C. for 30 seconds, 72.degree. C. for 4 minutes), 72.degree. C. for
7 minutes. The PCR reactions were separated on an agarose gel using
a Qiagen spin column. The fragments were then digested with BglII
and HindIII and purified using a Qiagen spin column. The purified
fragments were then ligated to BglII and HindIII digested pANT849
plasmids. Screening of mutants was performed in S. lividans and S.
rimosus.
8 Table of Characterized Mutants Mutant Position Substitution
Wild-type ebh24-16 92 Valine Isoleucine 237 Alanine Phenylalanine
ebh25-1 195 Serine Asparagine 294 Proline Serine ebh10-53 190
Tyrosine Phenylalanine 231 Arginine Glutamic acid ebh24-16d8 92
Valine Isoleucine 237 Alanine Phenylalanine 67 Glutamine Arginine
ebh24-16c11 92 Valine Isoleucine 93 Glycine Alanine 237 Alanine
Phenylalanine 365 Threonine Isoleucine ebh24-16-16 92 Valine
Isoleucine 106 Alanine Valine 237 Alanine Phenylalanine ebh24-16-74
88 Histidine Arginine 92 Valine Isoleucine 237 Alanine
Phenylalanine ebh-M18 31 Lysine Glutamic acid 176 Valine Methionine
ebh24-16g8 92 Valine Isoleucine 237 Alanine Phenylalanine 67
Glutamine Arginine 130 Threonine Isoleucine 176 Alanine Methionine
ebh24-16b9 92 Valine Isoleucine 237 Alanine Phenylalanine 67
Glutamine Arginine 140 Threonine Alanine 176 Serine Methionine
Example 18
[0244] Comparison of Epothilone B Transformation in Cells
Expressing ebh and Mutants Thereof
[0245] In these experiments, twenty ml of YEME medium in a 125 ml
bi-indented flask was inoculated with 200 .mu.l of a frozen spore
preparation of S. lividans TK24, S. lividans (pANT849-ebh), S.
lividans (pANT849-ebh10-53) or S. lividans (pANT849-ebh24-16) and
incubated 48 hours at 230 rpm, 30.degree. C. Thiostrepton, 10
.mu.g/ml was added to media inoculated with S. lividans
(pANT849-ebh), S. lividans (pANT849-ebh10-53) and S. lividans (pANT
849-ebh24-16). Four ml of culture was transferred to 20 ml of
R5medium in a 125 ml Erlenmeyer flask and incubated 18 hrs at 230
rpm, 30.degree. C. Epothilone B in 100% EtOH was added to each
culture to a final concentration of 0.05% weight/volume. Samples
were taken at 0, 24, 48 and 72 hours with the exception of the S.
lividans (pANT849-ebh24-16) culture, in which the epothilone B had
been completely converted to epothilone F at 48 hours. The samples
were analyzed by HPLC. Results were calculated as a percentage of
the epothilone B at time 0.
9 Epothilone B: Time pANT849- pANT849- (hours) TK24 pANT849-ebh
ebh10-53 ebh24-16 0 100% 100% 100% 100% 24 99% 78% 69% 56% 48 87%
19% 39% 0% 72 87% 0% 3% --
[0246]
10 Epothilone F: Time pANT849- pANT849- (hours) TK24 pANT849-ebh
ebh10-53 ebh24-16 0 0% 0% 0% 0% 24 0% 4% 9% 23% 48 0% 21% 29% 52%
72 0% 14% 41% --
[0247] Alternatively, the bioconversion of epothilone B to
epothilone F was performed in S. rimosus host cells transformed
with expression plasmids containing the ebh gene and its variants
or mutants. One-hundred .mu.l of a frozen S. rimosus transformant
culture was inoculated to 20 ml CRM media with 10 .mu.g/ml
thiostrepton and cultivated 16-24 hr, 30.degree. C., 230-300 rpm.
Epothilone B in 100% ethanol was added to each culture to a final
concentration of 0.05% weight/volume. The reaction was typically
incubated 20-40 hrs at 30.degree. C., 230-300 rpm. The
concentration of epothilones B and F was determined by HPLC
analysis.
11 Evaluation of mutants in S. rinosus Mutant Epothilone F yield
ebh-M18 55% ebh24-16d8 75% ebh24-16c11 75% ebh24-16-16 75%
ebh24-16-74 75% ebh24-16b9 80% ebh24-16g8 85%
Example 19
[0248] Biotransformation of Compactin to Pravastatin
[0249] Twenty ml of R2YE media with 10 .mu.g/ml thiostrepton in a
125 ml flask was inoculated with 200 .mu.l of a frozen spore
preparation of S. lividans (pANT849), S. lividans (pANT849-ebh) and
incubated 72 hours at 230 rpm, 28.degree. C. Four ml of culture was
inoculated to 20 ml of R2YE media and grown 24 hours at 230 rpm,
28.degree. C. One ml of culture was transferred to a 15 ml
polypropylene culture tube, 10 .mu.l of compactin (40 mg/ml) was
added to each culture and incubated for 24 hours, 28.degree. C.,
250 rpm. Five hundred .mu.l of the culture broth was transferred to
a fresh 15 ml polypropylene culture tube. Five hundred .mu.l of 50
mM sodium hydroxide was added and vortexed. Three ml of methanol
was added and vortexed, the tube was centrifuged 10 minutes at 3000
rpm in a TJ-6 table-top centrifuge. The organic phase was analyzed
by HPLC. Compactin and pravastatin values were assessed relative to
the control S. lividans (pANT849) culture.
[0250] Compactin and Pravastatin as a Percentage of Starting
Compactin Concentration:
12 S. lividans (pANT849) S. lividans (pANT849-ebh) Compactin 36%
11% Pravastatin 11% 53%
Example 20
[0251] High Performance Liquid Chromatography Method for Compactin
and Pravastatin Detection
[0252] The liquid chromatography separation was performed using a
Hewlett Packard1090 Series Separation system (Agilent Technologies,
Palo Alto, Calif., USA) and a column, 50.times.46 mm, filled with
Spherisorb ODS2, particle size 5 .mu.m (Keystone Scientific, Inc,
Bellefonte, Penn., USA). The gradient mobile phase programming was
used with a flow rate of 2.0 ml/minute. Eluent A was water, 10 mM
ammonium acetate and 0.05% Phosphoric Acid. Eluent B was
acetonitrile. The mobile phase was a linear gradient from 20% B to
90% B over 4 minutes.
Example 21
[0253] Structure Determination of the Biotransformation Product of
Mutant ebh25-1
[0254] Analytical HPLC was performed using a Hewlett Packard 1100
Series Liquid Chromatograph with a YMC Packed ODS-AQ column, 4.6 mm
i.d..times.15 cm 1. A gradient system of water (solvent A) and
acetonitrile (solvent B) was used: 20% to 90% B linear gradient, 10
minutes; 90% to 20% linear gradient, 2 minutes. The flow rate was 1
ml/minute and UV detection was at 254 nm.
[0255] Preparative HPLC was performed using the following equipment
and conditions:
[0256] Pump: Varian ProStar Solvent Delivery Module (Varian Inc.,
Palo Alto, Calif., USA). Detector: Gynkotek UVD340S.
[0257] Column: YMC ODS-A column (30 mmID.times.100 mm length, 5.mu.
.quadrature.particle size).
[0258] Elution flow rate: 30 ml/minute
[0259] Elution gradient: (solvent A: water; solvent B:
acetonitrile), 20% B, 2 minutes; 20% to 60% B linear gradient, 18
minutes; 60% B, 2 minutes; 60% to 90% B linear gradient, 1 minute;
90% B, 3 minutes; 90% to 20% B linear gradient, 2 minutes.
[0260] Detection: UV, 210 nm.
[0261] LC/NMR was performed as follows: 40 .mu.l of sample was
injected onto a YMC Packed ODS-AQ column (4.6 mm i.d..times.15 cm
1). The column was eluted at 1 ml/minute flow rate with a gradient
system of D.sub.2O (solvent A) and acetonitrile-d.sub.3 (solvent
B): 30% B, 1 minute; 30% to 80% B linear gradient, 11 minutes. The
eluent passed a UV detection cell (monitored at 254 nm) before
flowing through a F19/H1 NMR probe (60 .mu.l active volume) in
Varian AS-600 NMR spectrometer. The biotransformation product was
eluted at around 7.5 minutes and the flow was stopped manually to
allow the eluent to remain in the NMR probe for NMR data
acquisition.
[0262] Isolation and analysis was performed as follows. The
butanol/methanol extract (about 10 ml) was evaporated to dryness
under nitrogen stream. One ml methanol was added to the residue (38
mg) and insoluble material was removed by centrifugation (13000
rpm, 2 min). 0.1 ml of the supernatant was used for LC/NMR study
and the rest of 0.9 ml was subjected to the preparative HPLC
(0.2-0.4 ml per injection). Two major peaks were observed and
collected: peak A was eluted between 14 and 15 minutes, while peak
B was eluted between 16.5 and 17.5 minutes. Analytical HPLC
analysis indicated that peak B was the parent compound, epothilone
B (Rt 8.5 minutes), and peak A was the biotransformation product
(Rt 7.3 minutes). The peak A fractions were pooled and MS analysis
data was obtained with the pooled fractions. The pooled fraction
was evaporated to a small volume, then was lyophilized to give 3 mg
of white solid. NMR and HPLC analysis of the white solid (dissolved
in methanol) revealed that the biotransformation product was
partially decomposed during the drying process.
Sequence CWU 1
1
76 1 1186 DNA Amycolatopsis orientalis 1 atgaccgacg tcgaggaaac
caccgcgacc ttgccactgg cccgcaaatg cccgttttca 60 ccaccgcccg
aatacgagcg gctccgccgg gaaagtccgg tttcccgggt cggtctcccc 120
tccggtcaaa ccgcttgggc gctcacccgg ctcgaagaca tccgcgaaat gctgagcagt
180 ccgcatttca gctccgaccg gcagagtccg tcgttcccgc tgatggtggc
gcggcagatc 240 cggcgcgagg acaagccgtt ccgcccgtcc ctcatcgcga
tggacccgcc ggaacacggc 300 aaggccaggc gtgacgtcgt cggggaattc
accgtcaagc gcatgaaagc gcttcagcca 360 cgtattcagc agatcgtcga
cgagcatatc gacgccctgc tcgccggccc caaacccgcc 420 gatctcgtcc
aggcgctttc cctgccggtt ccgtccttgg tgatctgcga actgctcggt 480
gtcccctatt cggaccacga gttcttccag tcctgcagtt cccggatgct cagccgggaa
540 gtcaccgccg aagaacggat gaccgcgttc gagtcgctcg agaactatct
cgacgaactc 600 gtcacgaaga aggaggcgaa cgccaccgag gacgacctcc
tcggccgcca gatcctgaag 660 cagcgcgaat ccggcgaagc cgaccacggc
gaactggtcg gtctggcgtt cctcctgctc 720 atcgcggggc acgagactac
ggcgaacatg atctcgctcg gcacggtgac cctgctggag 780 aaccccgatc
agctggcgaa gatcaaggcg gatccgggca agaccctcgc cgcgatcgag 840
gaactcctgc ggatcttcac catcgcggag acggcgacct cacgcttcgc cacggcggac
900 gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg tcgtcggcct
gagcaacgcg 960 ggcaaccacg atccggacgg cttcgagaac ccggacacct
tcgacatcga acgcggcgcg 1020 cggcatcacg tcgccttcgg attcggtgtg
caccaatgcc tcggccagaa cttggcgagg 1080 ttggaactcc agatcgtgtt
cgatacgttg ttccggcgag tgccgggcat ccggatcgcc 1140 gtaccggtcg
acgaactgcc gttcaagcac gattcgacga tctacg 1186 2 404 PRT
Amycolatopsis orientalis 2 Met Thr Asp Val Glu Glu Thr Thr Ala Thr
Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro Phe Ser Pro Pro Pro Glu
Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30 Pro Val Ser Arg Val Gly
Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35 40 45 Thr Arg Leu Glu
Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe Ser 50 55 60 Ser Asp
Arg Gln Ser Pro Ser Phe Pro Leu Met Val Ala Arg Gln Ile 65 70 75 80
Arg Arg Glu Asp Lys Pro Phe Arg Pro Ser Leu Ile Ala Met Asp Pro 85
90 95 Pro Glu His Gly Lys Ala Arg Arg Asp Val Val Gly Glu Phe Thr
Val 100 105 110 Lys Arg Met Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile
Val Asp Glu 115 120 125 His Ile Asp Ala Leu Leu Ala Gly Pro Lys Pro
Ala Asp Leu Val Gln 130 135 140 Ala Leu Ser Leu Pro Val Pro Ser Leu
Val Ile Cys Glu Leu Leu Gly 145 150 155 160 Val Pro Tyr Ser Asp His
Glu Phe Phe Gln Ser Cys Ser Ser Arg Met 165 170 175 Leu Ser Arg Glu
Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu Ser 180 185 190 Leu Glu
Asn Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu Ala Asn Ala 195 200 205
Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu Lys Gln Arg Glu Ser 210
215 220 Gly Glu Ala Asp His Gly Glu Leu Val Gly Leu Ala Phe Leu Leu
Leu 225 230 235 240 Ile Ala Gly His Glu Thr Thr Ala Asn Met Ile Ser
Leu Gly Thr Val 245 250 255 Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala
Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys Thr Leu Ala Ala Ile Glu
Glu Leu Leu Arg Ile Phe Thr Ile 275 280 285 Ala Glu Thr Ala Thr Ser
Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290 295 300 Gly Thr Leu Ile
Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn Ala 305 310 315 320 Gly
Asn His Asp Pro Asp Gly Phe Glu Asn Pro Asp Thr Phe Asp Ile 325 330
335 Glu Arg Gly Ala Arg His His Val Ala Phe Gly Phe Gly Val His Gln
340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg Leu Glu Leu Gln Ile Val
Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val Pro Gly Ile Arg Ile Ala
Val Pro Val Asp 370 375 380 Glu Leu Pro Phe Lys His Asp Ser Thr Ile
Tyr Gly Leu His Ala Leu 385 390 395 400 Pro Val Thr Trp 3 195 DNA
Amycolatopsis orientalis 3 atgaagatca tcgcggacac cgggaagtgc
gtgggggcgg gccagtgcgt gctcaccgat 60 cccgatctgt tcgaccagag
cgaggacgac gggacggtcc tcctgctgaa cgccgagccc 120 gaaggcgaag
aggcggagga gaacgcgcgc accgccgtgc acatctgccc ggggcaggca 180
ctttcgctcg cgtag 195 4 64 PRT Amycolatopsis orientalis 4 Met Lys
Ile Ile Ala Asp Thr Gly Lys Cys Val Gly Ala Gly Gln Cys 1 5 10 15
Val Leu Thr Asp Pro Asp Leu Phe Asp Gln Ser Glu Asp Asp Gly Thr 20
25 30 Val Leu Leu Leu Asn Ala Glu Pro Glu Gly Glu Glu Ala Glu Glu
Asn 35 40 45 Ala Arg Thr Ala Val His Ile Cys Pro Gly Gln Ala Leu
Ser Leu Ala 50 55 60 5 22 DNA Artificial sequence Synthetic 5
tcctcatcgc cggccacgag ac 22 6 22 DNA Artificial sequence Synthetic
6 tgctggtcgc cggccacgag ac 22 7 22 DNA Artificial sequence
Synthetic 7 tgctcatcac cggccaggac ac 22 8 20 DNA Artificial
sequence Synthetic 8 ctgttcgccg ggcacgactc 20 9 22 DNA Artificial
sequence Synthetic 9 tgctcatcgc gggccacgag ac 22 10 22 DNA
Artificial sequence Synthetic 10 tgctggtcgc cgggcacgag ac 22 11 21
DNA Artificial sequence Synthetic 11 cggcgcggtg gaggaactgc t 21 12
21 DNA Artificial sequence Synthetic 12 gggcgccgtc gaggagctgc t 21
13 21 DNA Artificial sequence Synthetic 13 ccgcaccctg gaggagctgc t
21 14 21 DNA Artificial sequence Synthetic 14 cggcgcggtc gaggagatgc
t 21 15 21 DNA Artificial sequence Synthetic 15 cgcggcggtg
gaggagatgc t 21 16 21 DNA Artificial sequence Synthetic 16
cggcgcgatc gaggagaccc t 21 17 30 DNA Artificial sequence Synthetic
17 ttcggcttcg gcgtgcacca gtgcctgggc 30 18 30 DNA Artificial
sequence Synthetic 18 ttcggcttcg gcgtccacca gtgcctggga 30 19 30 DNA
Artificial sequence Synthetic 19 ttcggctggg gcccccacca ctgcctgggc
30 20 30 DNA Artificial sequence Synthetic 20 ttcggtcacg gcgtccacaa
gtgtcctggc 30 21 30 DNA Artificial sequence Synthetic 21 ttcgggcacg
gagcgcacca ctgcatcggc 30 22 30 DNA Artificial sequence Synthetic 22
ttcggccacg gcatccactt ctgcgtgggc 30 23 25 DNA Artificial sequence
Synthetic 23 tgctgctsdt cgccggbcab gasac 25 24 25 DNA Artificial
sequence Synthetic 24 tgmtssysnt cgscgsbcay gasac 25 25 24 DNA
Artificial sequence Synthetic 25 cggvgcsvts gaggarmtgc tgcg 24 26
24 DNA Artificial sequence Synthetic 26 cgcagcakyt cctcsabsgc bccg
24 27 30 DNA Artificial sequence Synthetic 27 gcccaggcas ahcacsyvvg
gcdybggctt 30 28 27 DNA Artificial sequence Synthetic 28 gcgagatcta
cctggggaag gacaacc 27 29 27 DNA Artificial sequence Synthetic 29
gcgaagctta cggacttgga ccctacg 27 30 1215 DNA Artificial sequence
Synthetic 30 atgaccgacg tcgaggaaac caccgcgacc ttgccactgg cccgcaaatg
cccgttttca 60 ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg
tttcccgggt cggtctcccc 120 tccggtcaaa ccgcttgggc gctcacccgg
ctcgaagaca tccgcgaaat gctgagcagt 180 ccgcatttca gctccgaccg
gcagagtccg tcgttcccgc tgatggtggc gcggcagatc 240 cggcgcgagg
acaagccgtt ccgcccgtcc ctcatcgcga tggacccgcc ggaacacggc 300
aaggccaggc gtgacgtcgt cggggaattc accgtcaagc gcatgaaagc gcttcagcca
360 cgtattcagc agatcgtcga cgagcatatc gacgccctgc tcgccggccc
caaacccgcc 420 gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg
tgatctgcga actgctcggt 480 gtcccctatt cggaccacga gttcttccag
tcctgcagtt cccggatgct cagccgggaa 540 gtcaccgccg aagaacggat
gaccgcgttc gagtcgctcg agagctatct cgacgaactc 600 gtcacgaaga
aggaggcgaa cgccaccgag gacgacctcc tcggccgcca gatcctgaag 660
cagcgcgaat ccggcgaagc cgaccacggc gaactggtcg gtctggcgtt cttgctgctc
720 atcgcggggc acgagactac ggcgaacatg atctcgctcg gcacggtgac
cctgctggag 780 aaccccgatc agctggcgaa gatcaaggcg gatccgggca
agaccctcgc cgcgatcgag 840 gaactcctgc ggatcttcac catcgcggag
acggcgaccc cacgcttcgc cacggcggac 900 gtcgagatcg gcggcacgct
catccgcgcg ggtgaaggcg tcgtcggcct gagcaacgcg 960 ggcaaccacg
atccggacgg cttcgagaac ccggacacct tcgacatcga acgcggcgcg 1020
cggcatcacg tcgccttcgg attcggtgtg caccaatgcc tcggccagaa cttggcgagg
1080 ttggaactcc agatcgtgtt cgatacgttg ttccggcgag tgccgggcat
ccggatcgcc 1140 gtaccggtcg acgaactgcc gttcaagcac gattcgacga
tctacggcct ccacgccctg 1200 ccggtcacct ggtag 1215 31 404 PRT
Artificial sequence Synthetic 31 Met Thr Asp Val Glu Glu Thr Thr
Ala Thr Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro Phe Ser Pro Pro
Pro Glu Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30 Pro Val Ser Arg
Val Gly Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35 40 45 Thr Arg
Leu Glu Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe Ser 50 55 60
Ser Asp Arg Gln Ser Pro Ser Phe Pro Leu Met Val Ala Arg Gln Ile 65
70 75 80 Arg Arg Glu Asp Lys Pro Phe Arg Pro Ser Leu Ile Ala Met
Asp Pro 85 90 95 Pro Glu His Gly Lys Ala Arg Arg Asp Val Val Gly
Glu Phe Thr Val 100 105 110 Lys Arg Met Lys Ala Leu Gln Pro Arg Ile
Gln Gln Ile Val Asp Glu 115 120 125 His Ile Asp Ala Leu Leu Ala Gly
Pro Lys Pro Ala Asp Leu Val Gln 130 135 140 Ala Leu Ser Leu Pro Val
Pro Ser Leu Val Ile Cys Glu Leu Leu Gly 145 150 155 160 Val Pro Tyr
Ser Asp His Glu Phe Phe Gln Ser Cys Ser Ser Arg Met 165 170 175 Leu
Ser Arg Glu Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu Ser 180 185
190 Leu Glu Ser Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu Ala Asn Ala
195 200 205 Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu Lys Gln Arg
Glu Ser 210 215 220 Gly Glu Ala Asp His Gly Glu Leu Val Gly Leu Ala
Phe Leu Leu Leu 225 230 235 240 Ile Ala Gly His Glu Thr Thr Ala Asn
Met Ile Ser Leu Gly Thr Val 245 250 255 Thr Leu Leu Glu Asn Pro Asp
Gln Leu Ala Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys Thr Leu Ala
Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr Ile 275 280 285 Ala Glu Thr
Ala Thr Pro Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290 295 300 Gly
Thr Leu Ile Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn Ala 305 310
315 320 Gly Asn His Asp Pro Asp Gly Phe Glu Asn Pro Asp Thr Phe Asp
Ile 325 330 335 Glu Arg Gly Ala Arg His His Val Ala Phe Gly Phe Gly
Val His Gln 340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg Leu Glu Leu
Gln Ile Val Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val Pro Gly Ile
Arg Ile Ala Val Pro Val Asp 370 375 380 Glu Leu Pro Phe Lys His Asp
Ser Thr Ile Tyr Gly Leu His Ala Leu 385 390 395 400 Pro Val Thr Trp
32 1215 DNA Artificial sequence Synthetic 32 atgaccgacg tcgaggaaac
caccgcgacc ttgccactgg cccgcaaatg cccgttttca 60 ccaccgcccg
aatacgagcg gctccgccgg gaaagtccgg tttcccgggt cggtctcccc 120
tccggtcaaa ccgcttgggc gctcacccgg ctcgaagaca tccgcgaaat gctgagcagt
180 ccgcatttca gctccgaccg gcagagtccg tcgttcccgc tgatggtggc
gcggcagatc 240 cggcgcgagg acaagccgtt ccgcccgtcc ctcatcgcga
tggacccgcc ggaacacggc 300 aaggccaggc gtgacgtcgt cggggaattc
accgtcaagc gcatgaaagc gcttcagcca 360 cgtattcagc agatcgtcga
cgagcatatc gacgccctgc tcgccggccc caaacccgcc 420 gatctcgtcc
aggcgctttc cctgccggtt ccgtccttgg tgatctgcga actgctcggt 480
gtcccctatt cggaccacga gttcttccag tcctgcagtt cccggatgct cagccgggaa
540 gtcaccgccg aagaacggat gaccgcgtac gagtcgctcg agaactatct
cgacgaactc 600 gtcacgaaga aggaggcgaa cgccaccgag gacgacctcc
tcggccgcca gatcctgaag 660 cagcgcgaat ccggcgaagc cgaccacggc
cgcctggtcg gtctggcgtt cctcctgctc 720 atcgcggggc acgagactac
ggcgaacatg atctcgctcg gcacggtgac cctgctggag 780 aaccccgatc
agctggcgaa gatcaaggcg gatccgggca agaccctcgc cgcgatcgag 840
gaactcctgc ggatcttcac catcgcggag acggcgacct cacgcttcgc cacggcggac
900 gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg tcgtcggcct
gagcaacgcg 960 ggcaaccacg atccggacgg cttcgagaac ccggacacct
tcgacatcga acgcggcgcg 1020 cggcatcacg tcgccttcgg attcggtgtg
caccaatgcc tcggccagaa cttggcgagg 1080 ttggaactcc agatcgtgtt
cgatacgttg ttccggcgag tgccgggcat ccggatcgcc 1140 gtaccggtcg
acgaactgcc gttcaagcac gattcgacga tctacggcct ccacgccctg 1200
ccggtcacct ggtag 1215 33 404 PRT Artificial sequence Synthetic 33
Met Thr Asp Val Glu Glu Thr Thr Ala Thr Leu Pro Leu Ala Arg Lys 1 5
10 15 Cys Pro Phe Ser Pro Pro Pro Glu Tyr Glu Arg Leu Arg Arg Glu
Ser 20 25 30 Pro Val Ser Arg Val Gly Leu Pro Ser Gly Gln Thr Ala
Trp Ala Leu 35 40 45 Thr Arg Leu Glu Asp Ile Arg Glu Met Leu Ser
Ser Pro His Phe Ser 50 55 60 Ser Asp Arg Gln Ser Pro Ser Phe Pro
Leu Met Val Ala Arg Gln Ile 65 70 75 80 Arg Arg Glu Asp Lys Pro Phe
Arg Pro Ser Leu Ile Ala Met Asp Pro 85 90 95 Pro Glu His Gly Lys
Ala Arg Arg Asp Val Val Gly Glu Phe Thr Val 100 105 110 Lys Arg Met
Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile Val Asp Glu 115 120 125 His
Ile Asp Ala Leu Leu Ala Gly Pro Lys Pro Ala Asp Leu Val Gln 130 135
140 Ala Leu Ser Leu Pro Val Pro Ser Leu Val Ile Cys Glu Leu Leu Gly
145 150 155 160 Val Pro Tyr Ser Asp His Glu Phe Phe Gln Ser Cys Ser
Ser Arg Met 165 170 175 Leu Ser Arg Glu Val Thr Ala Glu Glu Arg Met
Thr Ala Tyr Glu Ser 180 185 190 Leu Glu Asn Tyr Leu Asp Glu Leu Val
Thr Lys Lys Glu Ala Asn Ala 195 200 205 Thr Glu Asp Asp Leu Leu Gly
Arg Gln Ile Leu Lys Gln Arg Glu Ser 210 215 220 Gly Glu Ala Asp His
Gly Arg Leu Val Gly Leu Ala Phe Leu Leu Leu 225 230 235 240 Ile Ala
Gly His Glu Thr Thr Ala Asn Met Ile Ser Leu Gly Thr Val 245 250 255
Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala Lys Ile Lys Ala Asp Pro 260
265 270 Gly Lys Thr Leu Ala Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr
Ile 275 280 285 Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr Ala Asp Val
Glu Ile Gly 290 295 300 Gly Thr Leu Ile Arg Ala Gly Glu Gly Val Val
Gly Leu Ser Asn Ala 305 310 315 320 Gly Asn His Asp Pro Asp Gly Phe
Glu Asn Pro Asp Thr Phe Asp Ile 325 330 335 Glu Arg Gly Ala Arg His
His Val Ala Phe Gly Phe Gly Val His Gln 340 345 350 Cys Leu Gly Gln
Asn Leu Ala Arg Leu Glu Leu Gln Ile Val Phe Asp 355 360 365 Thr Leu
Phe Arg Arg Val Pro Gly Ile Arg Ile Ala Val Pro Val Asp 370 375 380
Glu Leu Pro Phe Lys His Asp Ser Thr Ile Tyr Gly Leu His Ala Leu 385
390 395 400 Pro Val Thr Trp 34 1215 DNA Artificial sequence
Synthetic 34 atgaccgacg tcgaggaaac caccgcgacc ttgccactgg cccgcaaatg
cccgttttca 60 ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg
tttcccgggt cggtctcccc 120 tccggtcaaa ccgcttgggc gctcacccgg
ctcgaagaca tccgcgaaat gctgagcagt 180 ccgcatttca gctccgaccg
gcagagtccg tcgttcccgc tgatggtggc gcggcagatc 240 cggcgcgagg
acaagccgtt ccgcccgtcc ctcgtcgcga tggacccgcc ggaacacggc 300
aaggccaggc gtgacgtcgt cggggaattc accgtcaagc gcatgaaagc gcttcagcca
360 cgtattcagc agatcgtcga cgagcatatc gacgccctgc tcgccggccc
caaacccgcc 420 gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg
tgatctgcga actgctcggt 480 gtcccctatt cggaccacga gttcttccag
tcctgcagtt cccggatgct cagccgggaa 540
gtcaccgccg aagaacggat gaccgcgttc gagtcgctcg agaactatct cgacgaactc
600 gtcacgaaga aggaggcgaa cgccaccgag gacgacctcc tcggccgcca
gatcctgaag 660 cagcgcgaat ccggcgaagc cgaccacggc gaactggtcg
gtctggcggc gctcctgctc 720 atcgcggggc acgagactac ggcgaacatg
atctcgctcg gcacggtgac cctgctggag 780 aaccccgatc agctggcgaa
gatcaaggcg gatccgggca agaccctcgc cgcgatcgag 840 gaactcctgc
ggatcttcac catcgcggag acggcgacct cacgcttcgc cacggcggac 900
gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg tcgtcggcct gagcaacgcg
960 ggcaaccacg atccggacgg cttcgagaac ccggacacct tcgacatcga
acgcggcgcg 1020 cggcatcacg tcgccttcgg attcggtgtg caccaatgcc
tcggccagaa cttggcgagg 1080 ttggaactcc agatcgtgtt cgatacgttg
ttccggcgag tgccgggcat ccggatcgcc 1140 gtaccggtcg acgaactgcc
gttcaagcac gattcgacga tctacggcct ccacgccctg 1200 ccggtcacct ggtag
1215 35 404 PRT Artificial sequence Synthetic 35 Met Thr Asp Val
Glu Glu Thr Thr Ala Thr Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro
Phe Ser Pro Pro Pro Glu Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30
Pro Val Ser Arg Val Gly Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35
40 45 Thr Arg Leu Glu Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe
Ser 50 55 60 Ser Asp Arg Gln Ser Pro Ser Phe Pro Leu Met Val Ala
Arg Gln Ile 65 70 75 80 Arg Arg Glu Asp Lys Pro Phe Arg Pro Ser Leu
Val Ala Met Asp Pro 85 90 95 Pro Glu His Gly Lys Ala Arg Arg Asp
Val Val Gly Glu Phe Thr Val 100 105 110 Lys Arg Met Lys Ala Leu Gln
Pro Arg Ile Gln Gln Ile Val Asp Glu 115 120 125 His Ile Asp Ala Leu
Leu Ala Gly Pro Lys Pro Ala Asp Leu Val Gln 130 135 140 Ala Leu Ser
Leu Pro Val Pro Ser Leu Val Ile Cys Glu Leu Leu Gly 145 150 155 160
Val Pro Tyr Ser Asp His Glu Phe Phe Gln Ser Cys Ser Ser Arg Met 165
170 175 Leu Ser Arg Glu Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu
Ser 180 185 190 Leu Glu Asn Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu
Ala Asn Ala 195 200 205 Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu
Lys Gln Arg Glu Ser 210 215 220 Gly Glu Ala Asp His Gly Glu Leu Val
Gly Leu Ala Ala Leu Leu Leu 225 230 235 240 Ile Ala Gly His Glu Thr
Thr Ala Asn Met Ile Ser Leu Gly Thr Val 245 250 255 Thr Leu Leu Glu
Asn Pro Asp Gln Leu Ala Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys
Thr Leu Ala Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr Ile 275 280 285
Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290
295 300 Gly Thr Leu Ile Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn
Ala 305 310 315 320 Gly Asn His Asp Pro Asp Gly Phe Glu Asn Pro Asp
Thr Phe Asp Ile 325 330 335 Glu Arg Gly Ala Arg His His Val Ala Phe
Gly Phe Gly Val His Gln 340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg
Leu Glu Leu Gln Ile Val Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val
Pro Gly Ile Arg Ile Ala Val Pro Val Asp 370 375 380 Glu Leu Pro Phe
Lys His Asp Ser Thr Ile Tyr Gly Leu His Ala Leu 385 390 395 400 Pro
Val Thr Trp 36 1104 DNA Amycolatopsis orientalis 36 gcgaccttgc
cgctggcccg caaatgcccg ttttcaccgc cgcccgaata cgagcggctt 60
cgccgggaaa gtccggtttc ccgggtcggt ctcccgtccg gtcaaaccgc ttgggcgctc
120 acccggctcg aggacatccg cgaaatgctg agcagtccgc atttcagctc
cgaccggcag 180 agtccgtcgt tcccgctgat ggtggcccgg cagatccggc
gcgaggacaa gccgttccgc 240 ccgtccctca tcgcgatgga cccgccggaa
cacagcaagg ccaggcgtga cgtcgtcggg 300 gaattcaccg tcaagcgcat
gaaagcgctt cagccgcgta ttcagcagat cgtcgacgag 360 catatcgacg
ccatgctcgc cggccccaaa cccgccgatc tcgtccaggc gctttccctg 420
ccggttccgt ccttggtgat ctgcgaactg ctcggtgtcc cctattcgga ccacgagttc
480 ttccagtcct gcagttcccg gatgctcagc cgggaagtca ccgccgaaga
acggatgacc 540 gcgttcgagt cgctcgagaa ctatctcgac gaactcgtca
cgaagaagga ggcgaacgcc 600 accgaggacg acctcctcgg ccgccagatc
ctgaagcagc gcgaaacggg cgaagccgac 660 cacggcgaac tcgtcgggct
ggcgttcctg ctgctcatcg cgggacacga gacgacggcg 720 aacatgatct
cgctcggcac ggcgaccctg ctggagaacc ccgaccagct ggcgaagatc 780
aaggccgatc cgggcaagac cctcgccgcg atcgaggagc tcctgcgggt cttcaccatc
840 gcggagacgg cgacctcacg cttcgccacg gcggacgtcg agatcggcgg
cacgctcatc 900 cgcgcgggtg aaggcgtcgt cggcctgagc aacgcgggca
accacgatcc ggaaggcttc 960 gagaacccgg acgccttcga catcgaacgc
ggcgcgcggc accacgtcgc cttcggattc 1020 ggtgtgcacc aatgcctcgg
ccagaacttg gcgaggttgg aactccagat cgtgttcgat 1080 acgttgttcc
ggcgagtgcc gggc 1104 37 1103 DNA Amycolatopsis orientalis 37
gaccttgccg ctggcccgga aatgcccgtt ttcgccgccg cccgaatacg aacggcttcg
60 ccgggaaagt ccggtttccc gggtcggtct cccgtccggt caaacggctt
gggcgctcac 120 ccggctcgaa gacatccgcg aaatgctgag cagcccgcat
ttcagttccg accggcagag 180 cccgtcgttc ccgctgatgg tcgcgcggca
gatccgccgc gaggacaagc cgttccgccc 240 ctccctcatc gcgatggatc
cgccggaaca cagccgggcc aggcgtgacg tcgtcgggga 300 attcaccgtc
aagcggatga aggcgctcca gccgcgaatt cagcagatcg tcgacgaaca 360
tctcgacgcc ctgctcgcgg gccccaaacc cgccgatctc gtccaggcgc tttccctgcc
420 cgttccctcg ctggtgatct gcgaactgct cggcgtcccc tattcggacc
acgagttctt 480 ccagtcctgc agttccagga tgctcagccg ggaggtcacc
gccgaagaac ggatgaccgc 540 gttcgagcag ctcgaaaact atctcgacga
actggtcacc aagaaggagg cgaacgccac 600 cgaggacgac ctcctcggcc
gtcagatcct gaaacagcgg gaaacgggcg aggccgacca 660 cggtgaactc
gtcgggctgg cgttcctgct gctcatcgcc ggacacgaga ccacggcgaa 720
catgatctcg ctcggcacgg tgaccctgct ggagaatccc gatcagctcg cgaagatcaa
780 ggcagacccc ggcaagaccc tcgccgccat cgaggaactc ctgcgggtct
tcacgatcgc 840 ggaaacggcg acctcacgct tcgccacggc ggacgtcgag
atcggcggaa cgctgatccg 900 cgcgggggaa ggggtggtgg gcctgagcaa
cgcgggcaac cacgatccgg acggcttcga 960 gaacccggac accttcgaca
tcgaacgcgg cgcgcggcat cacgtcgcgt tcggattcgg 1020 ggtgcaccag
tgtctcggcc agaacttggc gaggttggaa ctccagatcg tcttcgatac 1080
gttgttccgg cgagtgccgg gcc 1103 38 817 DNA Amycolatopsis orientalis
38 cttcacccgc gcggatgagc gtgccgccga tctcgacgtc cgccgtggcg
aagcgtgagg 60 tcgccgtctc cgcgatggtg aagatccgca ggagttcctc
gatcgcggcg agggtcttgc 120 ccggatccgc cttgatcttc gccagctgat
cggggttctc cagcagggtc accgtgccga 180 gcgagatcat gttcgccgta
gtctcgtgcc ccgcgatgag caggaggaac gccagaccga 240 ccagttcgcc
gtggtcggct tcgccggatt cgcgctgctt caggatctgg cggccgagga 300
ggtcgtcctc ggtggcgttc gcctccttct tcgtgacgag ttcgtcgaga tagttctcga
360 gcgactcgaa cgcggtcatc cgttcttcgg cggtgacttc ccggctgagc
atccgggaac 420 tgcaggactg gaagaactcg tggtccgaat aggggacacc
gagcagttcg cagatcacca 480 aggacggaac cggcagggaa agcgcctgga
cgagatcggc gggtttgggg ccggcgagca 540 gggcgtcgat atgctcgtcg
acgatctgct gaatacgtgg ctgaagcgct ttcatgcgct 600 tgacggtgaa
ttccccgacg acgtcacgcc tggccttgcc gtgttccggc gggtccatcg 660
cgatgaggga cgggcggaac ggcttgtcct cgcgccggat ctgccgcgcc accatcagcg
720 ggaacgacgg actctgccgg tcggagctga aatgcggact gctcagcatt
tcgcggatgt 780 cttcgagccg ggtgagcgcc caagcggttt gaccgga 817 39 1105
DNA Amycolatopsis orientalis 39 ccgcgacctt gccgctggcc cgcaaatgcc
cgttttcacc gccgcccgaa tacgagcggc 60 ttcgccggga aagtccggtt
tcccgggtcg gtctcccgtc cggtcaaacc gcttgggcgc 120 tcacccggct
cgaggacatc cgcgaaatgc tgagcagtcc gcatttcagc tccgaccggc 180
agagtccgtc gttcccgctg atggtggccc ggcagatccg gcgcgaggac aagccgttcc
240 gcccgtccct catctcgatg gacccgccgg aacacagcaa ggccaggcgt
gacgtcgtcg 300 gggaattcac cgtcaagcgc atgaaagcgc ttcagccgcg
tattcagcag atcgtcgacg 360 agcatatcga cgccctgctc gccggcccca
aacccgccga tctcgtccag gcgctttccc 420 tgccggttcc gtccttggtg
atctgcgaac tgctcggtgt cccctattcg gaccacgagt 480 tcttccagtc
ctgcagttcc cggatgctca gccgggaagt caccgccgaa gaacggatga 540
ccgcgttcga gtcgctcgag aactatctcg acgaactcgt cacgaagaag gaggcgaacg
600 ccaccgagga cgacctcctc ggccgccaga tcctgaagca gcgcgaaacg
ggcgaagccg 660 accacggcga actggtcggg ctggcgttcc tcctgctcat
cgcgggacac gagacgacgg 720 cgaacatgat ctcgctcggc acggcgaccc
tgctggagaa ccccgaccag ctggcgaaga 780 tcaaggccga tccgggcaag
accctcgccg cgatcgagga gctcctgcgg gtcttcacca 840 tcgcggagac
ggcgacctca cgcttcgcca cggcggacgt cgagatcggc ggcacgctca 900
tccgcgcggg tgaaggcgtc gtcggcctga gtaacgcggg caaccacgat ccggaaggct
960 tcgagaaccc ggacgccttc gacatcgaac gcggcgcgcg gcaccacgtc
gccttcggat 1020 tcggtgtgca ccaatgcctc ggccagaact tggcgaggtt
ggaactccag atcgtgttcg 1080 atacgttgtt ccggcgagtg ccggg 1105 40 1304
DNA Amycolatopsis orientalis 40 ccttgccact ggcccgcaaa tgcccgtttt
caccaccgcc cgaatacgag cggctccgcc 60 gggaaagtcc ggtttcccgg
gtcggtctcc cctccggtca aaccgcttgg gcgctcaccc 120 ggctcgaaga
catccgcgaa atgctgagca gtccgcattt cagctccgac cggcagagtc 180
cgtcgttccc gctgatggtg gcgcggcaga tccggcgcga ggacaagccg ttccgcccgt
240 ccctcatcgc gatggacccg ccggaacacg gcaaggccag gcgtgacgtc
gtcggggaat 300 tcaccgtcaa gcgcatgaaa gcgcttcagc cacgtattca
gcagatcgtc gacgagcata 360 tcgacgccct gctcgccggc cccaaacccg
ccgatctcgt ccaggcgctt tccctgccgg 420 ttccgtcctt ggtgatctgc
gaactgctcg gtgtccccta ttcggaccac gagttcttcc 480 agtcctgcag
ttcccggatg ctcagccggg aagtcaccgc cgaagaacgg atgaccgcgt 540
tcgagtcgct cgagaactat ctcgacgaac tcgtcacgaa gaaggaggcg aacgccaccg
600 aggacgacct cctcggccgc cagatcctga agcagcgcga atccggcgaa
gccgaccacg 660 gcgaactggt cggtctggcg ttcctcctgc tcatcgcggg
gcacgagact acggcgaaca 720 tgatctcgct cggcacggtg accctgctgg
agaaccccga tcagctggcg aagatcaagg 780 cggatccggg caagaccctc
gccgcgatcg aggaactcct gcggatcttc accatcgcgg 840 agacggcgac
ctcacgcttc gccacggcgg acgtcgagat cggcggcacg ctcatccgcg 900
cgggtgaagg cgtcgtcggc ctgagcaacg cgggcaacca cgatccggac ggcttcgaga
960 acccggacac cttcgacatc gaacgcggcg cgcggcatca cgtcgccttc
ggattcggtg 1020 tgcaccaatg cctcggccag aacttggcga ggttggaact
ccagatcgtg ttcgatacgt 1080 tgttccggcg agtgccgggc atccggatcg
ccgtaccggt cgacgaactg ccgttcaagc 1140 acgattcgac gatctacggc
ctccgcgccc tgccggtcac ctggtaggag gagccatgaa 1200 gatcatcgcg
gacaccggga agtgcgtggg ggcgggccag tgcgtgctca ccgatcccga 1260
tctgttcgac cagagcgagg acgacgggac ggtcctcctg ctga 1304 41 825 DNA
Amycolatopsis orientalis 41 ctccggtcaa accgcttggg cgctcacccg
gctcgaagac atccgcgaaa tgctgagcag 60 tccgcatttc agctccgacc
ggcagaatcc gtcgttcccg ctgatggtgg cgcggcagat 120 ccggcgcgag
gacaagccgt tccgcccgtc cctcatcgcg atggacccgc cggaacacag 180
caaggccagg cgtgacgtcg tcggggaatt caccgtcaag cgcatgaaag cgcttcagcc
240 gcgtattcag cagatcgtcg acgagcatat cgacgccctg ctcgccggcc
ccaaacccgc 300 cgatctcgtc caggcgcttt ccctgccggt tccgtccttg
gtgatctgcg aactgctcgg 360 tgtcccctat tcggaccacg agttcttcca
gtcctgcagt tcccggatgc tcagccggga 420 agtcaccgcc gaagaacgga
tgaccgcgtt cgagtcgctc gagaactatc tcgacgaact 480 cgtcacgaag
aaggaggcga acgccaccga ggacgacctc ctcggccgcc agatcctgaa 540
gcagcgggaa acgggcgagg ccgaccacgg cgaactcgtc gggctggcgt tcctgctgct
600 catcgccggg cacgagacga cggcgaacat gatctcgctc ggcacggcga
ccctgctgga 660 gaaccccgac cagctggcga agatcaaggc ggatccgggc
aagaccctcg ccgcgatcga 720 ggaactgctg cgcgtcttca cgatcgcgga
gacggcgacc tcacgcttcg ccacggcgga 780 cgtcgagatc ggcggcacgc
tcatccgcgc gggtgaaggc gtcgt 825 42 1103 DNA Amycolatopsis
orientalis 42 gcgaccttgc cactggcccg caaatgcccg ttttcaccac
cgcccgaata cgagcggctc 60 cgccgggaaa gtccggtttc ccgggtcggt
ctcccctccg gtcaaaccgc ttgggcgctc 120 acccggctcg aagacatccg
cgaaatgctg agcagtccgc atttcagctc cgaccggcag 180 agtccgtcgt
tcccgctgat ggtggcgcgg cagatccggc gcgaggacaa gccgttccgc 240
ccgtccctca tcgcgatgga cccgccggaa cacggcaagg ccaggcgtga cgtcgtcggg
300 gaattcaccg tcaagcgcat gaaagcgctt cagccacgta ttcagcagat
cgtcgacgag 360 catatcgacg ccctgctcgc cggccccaaa cccgccgatc
tcgtccaggc gctttccctg 420 ccggttccgt ccttggtgat ctgcgaactg
ctcggtgtcc cctattcgga ccacgagttc 480 ttccagtcct gcagttcccg
gatgctcagc cgggaagtca ccgccgaaga acggatgacc 540 gcgttcgagt
cgctcgagaa ctatctcgac gaactcgtca cgaagaagga ggcgaacgcc 600
accgaggacg acctcctcgg ccgccagatc ctgaagcagc gcgaatccgg cgaagccgac
660 cacggcgaac tggtcggtct ggcgttcctc ctgctcatcg cggggcacga
gactacggcg 720 aacatgatct cgctcggcac ggtgaccctg ctggagaacc
ccgatcagct ggcgaagatc 780 aaggcggatc cgggcaagac cctcgccgcg
atcgaggaac tcctgcggat cttcaccatc 840 gcggagacgg cgacctcacg
cttcgccacg gcggacgtcg agatcggcgg cacgctcatc 900 cgcgcgggtg
aaggcgtcgt cggcctgagc aacgcgggca accacgatcc ggacggcttc 960
gagaacccgg acaccttcga catcgaacgc ggcgcgcggc atcacgtcgc cttcggattc
1020 ggtgtgcacc aatgcctcgg ccagaacttg gcgaggttgg aactccagat
cgtgttcgat 1080 acgttgttcc ggcgagtgcc ggg 1103 43 402 PRT
Amycolatopsis orientalis 43 Met Thr Asp Val Glu Glu Thr Thr Ala Thr
Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro Phe Ser Pro Pro Pro Glu
Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30 Pro Val Ser Arg Val Gly
Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35 40 45 Thr Arg Leu Glu
Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe Ser 50 55 60 Ser Asp
Arg Gln Ser Pro Ser Phe Pro Leu Met Val Ala Arg Gln Ile 65 70 75 80
Arg Arg Glu Asp Lys Pro Phe Arg Pro Ser Leu Ile Ala Met Asp Pro 85
90 95 Pro Glu His Ser Lys Ala Arg Arg Asp Val Val Gly Glu Phe Thr
Val 100 105 110 Lys Arg Met Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile
Val Asp Glu 115 120 125 His Ile Asp Ala Met Leu Ala Gly Pro Lys Pro
Ala Asp Leu Val Gln 130 135 140 Ala Leu Ser Leu Pro Val Pro Ser Leu
Val Ile Cys Glu Leu Leu Gly 145 150 155 160 Val Pro Tyr Ser Asp His
Glu Phe Phe Gln Ser Cys Ser Ser Arg Met 165 170 175 Leu Ser Arg Glu
Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu Ser 180 185 190 Leu Glu
Asn Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu Ala Asn Ala 195 200 205
Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu Lys Gln Arg Glu Thr 210
215 220 Gly Glu Ala Asp His Gly Glu Leu Val Gly Leu Ala Phe Leu Leu
Leu 225 230 235 240 Ile Ala Gly His Glu Thr Thr Ala Asn Met Ile Ser
Leu Gly Thr Ala 245 250 255 Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala
Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys Thr Leu Ala Ala Ile Glu
Glu Leu Leu Arg Val Phe Thr Ile 275 280 285 Ala Glu Thr Ala Thr Ser
Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290 295 300 Gly Thr Leu Ile
Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn Ala 305 310 315 320 Gly
Asn His Asp Pro Glu Gly Phe Glu Asn Pro Asp Ala Phe Asp Ile 325 330
335 Glu Arg Gly Ala Arg His His Val Ala Phe Gly Phe Gly Val His Gln
340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg Leu Glu Leu Gln Ile Val
Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val Pro Gly Ile Arg Ile Ala
Val Pro Val Asp 370 375 380 Glu Leu Pro Phe Lys His Asp Ser Thr Ile
Tyr Gly Leu His Ala Leu 385 390 395 400 Pro Val 44 367 PRT
Amycolatopsis orientalis 44 Thr Leu Pro Leu Ala Arg Lys Cys Pro Phe
Ser Pro Pro Pro Glu Tyr 1 5 10 15 Glu Arg Leu Arg Arg Glu Ser Pro
Val Ser Arg Val Gly Leu Pro Ser 20 25 30 Gly Gln Thr Ala Trp Ala
Leu Thr Arg Leu Glu Asp Ile Arg Glu Met 35 40 45 Leu Ser Ser Pro
His Phe Ser Ser Asp Arg Gln Ser Pro Ser Phe Pro 50 55 60 Leu Met
Val Ala Arg Gln Ile Arg Arg Glu Asp Lys Pro Phe Arg Pro 65 70 75 80
Ser Leu Ile Ala Met Asp Pro Pro Glu His Ser Arg Ala Arg Arg Asp 85
90 95 Val Val Gly Glu Phe Thr Val Lys Arg Met Lys Ala Leu Gln Pro
Arg 100 105 110 Ile Gln Gln Ile Val Asp Glu His Leu Asp Ala Leu Leu
Ala Gly Pro 115 120 125 Lys Pro Ala Asp Leu Val Gln Ala Leu Ser Leu
Pro Val Pro Ser Leu 130 135 140 Val Ile Cys Glu Leu Leu Gly Val Pro
Tyr Ser Asp His Glu Phe Phe 145 150 155 160 Gln Ser Cys Ser Ser Arg
Met Leu Ser Arg Glu Val Thr Ala Glu Glu 165 170 175 Arg Met Thr Ala
Phe Glu Gln Leu Glu Asn Tyr Leu Asp Glu Leu Val 180 185 190 Thr Lys
Lys Glu Ala Asn Ala Thr Glu Asp Asp Leu Leu Gly Arg Gln 195 200 205
Ile Leu Lys Gln Arg Glu Thr Gly Glu Ala Asp His Gly Glu Leu Val 210
215 220 Gly Leu Ala Phe Leu Leu Leu Ile Ala Gly His Glu Thr Thr Ala
Asn 225 230 235 240 Met Ile
Ser Leu Gly Thr Val Thr Leu Leu Glu Asn Pro Asp Gln Leu 245 250 255
Ala Lys Ile Lys Ala Asp Pro Gly Lys Thr Leu Ala Ala Ile Glu Glu 260
265 270 Leu Leu Arg Val Phe Thr Ile Ala Glu Thr Ala Thr Ser Arg Phe
Ala 275 280 285 Thr Ala Asp Val Glu Ile Gly Gly Thr Leu Ile Arg Ala
Gly Glu Gly 290 295 300 Val Val Gly Leu Ser Asn Ala Gly Asn His Asp
Pro Asp Gly Phe Glu 305 310 315 320 Asn Pro Asp Thr Phe Asp Ile Glu
Arg Gly Ala Arg His His Val Ala 325 330 335 Phe Gly Phe Gly Val His
Gln Cys Leu Gly Gln Asn Leu Ala Arg Leu 340 345 350 Glu Leu Gln Ile
Val Phe Asp Thr Leu Phe Arg Arg Val Pro Gly 355 360 365 45 272 PRT
Amycolatopsis orientalis 45 Ser Gly Gln Thr Ala Trp Ala Leu Thr Arg
Leu Glu Asp Ile Arg Glu 1 5 10 15 Met Leu Ser Ser Pro His Phe Ser
Ser Asp Arg Gln Ser Pro Ser Phe 20 25 30 Pro Leu Met Val Ala Arg
Gln Ile Arg Arg Glu Asp Lys Pro Phe Arg 35 40 45 Pro Ser Leu Ile
Ala Met Asp Pro Pro Glu His Gly Lys Ala Arg Arg 50 55 60 Asp Val
Val Gly Glu Phe Thr Val Lys Arg Met Lys Ala Leu Gln Pro 65 70 75 80
Arg Ile Gln Gln Ile Val Asp Glu His Ile Asp Ala Leu Leu Ala Gly 85
90 95 Pro Lys Pro Ala Asp Leu Val Gln Ala Leu Ser Leu Pro Val Pro
Ser 100 105 110 Leu Val Ile Cys Glu Leu Leu Gly Val Pro Tyr Ser Asp
His Glu Phe 115 120 125 Phe Gln Ser Cys Ser Ser Arg Met Leu Ser Arg
Glu Val Thr Ala Glu 130 135 140 Glu Arg Met Thr Ala Phe Glu Ser Leu
Glu Asn Tyr Leu Asp Glu Leu 145 150 155 160 Val Thr Lys Lys Glu Ala
Asn Ala Thr Glu Asp Asp Leu Leu Gly Arg 165 170 175 Gln Ile Leu Lys
Gln Arg Glu Ser Gly Glu Ala Asp His Gly Glu Leu 180 185 190 Val Gly
Leu Ala Phe Leu Leu Leu Ile Ala Gly His Glu Thr Thr Ala 195 200 205
Asn Met Ile Ser Leu Gly Thr Val Thr Leu Leu Glu Asn Pro Asp Gln 210
215 220 Leu Ala Lys Ile Lys Ala Asp Pro Gly Lys Thr Leu Ala Ala Ile
Glu 225 230 235 240 Glu Leu Leu Arg Ile Phe Thr Ile Ala Glu Thr Ala
Thr Ser Arg Phe 245 250 255 Ala Thr Ala Asp Val Glu Ile Gly Gly Thr
Leu Ile Arg Ala Gly Glu 260 265 270 46 367 PRT Amycolatopsis
orientalis 46 Ala Thr Leu Pro Leu Ala Arg Lys Cys Pro Phe Ser Pro
Pro Pro Glu 1 5 10 15 Tyr Glu Arg Leu Arg Arg Glu Ser Pro Val Ser
Arg Val Gly Leu Pro 20 25 30 Ser Gly Gln Thr Ala Trp Ala Leu Thr
Arg Leu Glu Asp Ile Arg Glu 35 40 45 Met Leu Ser Ser Pro His Phe
Ser Ser Asp Arg Gln Ser Pro Ser Phe 50 55 60 Pro Leu Met Val Ala
Arg Gln Ile Arg Arg Glu Asp Lys Pro Phe Arg 65 70 75 80 Pro Ser Leu
Ile Ser Met Asp Pro Pro Glu His Ser Lys Ala Arg Arg 85 90 95 Asp
Val Val Gly Glu Phe Thr Val Lys Arg Met Lys Ala Leu Gln Pro 100 105
110 Arg Ile Gln Gln Ile Val Asp Glu His Ile Asp Ala Leu Leu Ala Gly
115 120 125 Pro Lys Pro Ala Asp Leu Val Gln Ala Leu Ser Leu Pro Val
Pro Ser 130 135 140 Leu Val Ile Cys Glu Leu Leu Gly Val Pro Tyr Ser
Asp His Glu Phe 145 150 155 160 Phe Gln Ser Cys Ser Ser Arg Met Leu
Ser Arg Glu Val Thr Ala Glu 165 170 175 Glu Arg Met Thr Ala Phe Glu
Ser Leu Glu Asn Tyr Leu Asp Glu Leu 180 185 190 Val Thr Lys Lys Glu
Ala Asn Ala Thr Glu Asp Asp Leu Leu Gly Arg 195 200 205 Gln Ile Leu
Lys Gln Arg Glu Thr Gly Glu Ala Asp His Gly Glu Leu 210 215 220 Val
Gly Leu Ala Phe Leu Leu Leu Ile Ala Gly His Glu Thr Thr Ala 225 230
235 240 Asn Met Ile Ser Leu Gly Thr Ala Thr Leu Leu Glu Asn Pro Asp
Gln 245 250 255 Leu Ala Lys Ile Lys Ala Asp Pro Gly Lys Thr Leu Ala
Ala Ile Glu 260 265 270 Glu Leu Leu Arg Val Phe Thr Ile Ala Glu Thr
Ala Thr Ser Arg Phe 275 280 285 Ala Thr Ala Asp Val Glu Ile Gly Gly
Thr Leu Ile Arg Ala Gly Glu 290 295 300 Gly Val Val Gly Leu Ser Asn
Ala Gly Asn His Asp Pro Glu Gly Phe 305 310 315 320 Glu Asn Pro Asp
Ala Phe Asp Ile Glu Arg Gly Ala Arg His His Val 325 330 335 Ala Phe
Gly Phe Gly Val His Gln Cys Leu Gly Gln Asn Leu Ala Arg 340 345 350
Leu Glu Leu Gln Ile Val Phe Asp Thr Leu Phe Arg Arg Val Pro 355 360
365 47 394 PRT Amycolatopsis orientalis 47 Leu Pro Leu Ala Arg Lys
Cys Pro Phe Ser Pro Pro Pro Glu Tyr Glu 1 5 10 15 Arg Leu Arg Arg
Glu Ser Pro Val Ser Arg Val Gly Leu Pro Ser Gly 20 25 30 Gln Thr
Ala Trp Ala Leu Thr Arg Leu Glu Asp Ile Arg Glu Met Leu 35 40 45
Ser Ser Pro His Phe Ser Ser Asp Arg Gln Ser Pro Ser Phe Pro Leu 50
55 60 Met Val Ala Arg Gln Ile Arg Arg Glu Asp Lys Pro Phe Arg Pro
Ser 65 70 75 80 Leu Ile Ala Met Asp Pro Pro Glu His Gly Lys Ala Arg
Arg Asp Val 85 90 95 Val Gly Glu Phe Thr Val Lys Arg Met Lys Ala
Leu Gln Pro Arg Ile 100 105 110 Gln Gln Ile Val Asp Glu His Ile Asp
Ala Leu Leu Ala Gly Pro Lys 115 120 125 Pro Ala Asp Leu Val Gln Ala
Leu Ser Leu Pro Val Pro Ser Leu Val 130 135 140 Ile Cys Glu Leu Leu
Gly Val Pro Tyr Ser Asp His Glu Phe Phe Gln 145 150 155 160 Ser Cys
Ser Ser Arg Met Leu Ser Arg Glu Val Thr Ala Glu Glu Arg 165 170 175
Met Thr Ala Phe Glu Ser Leu Glu Asn Tyr Leu Asp Glu Leu Val Thr 180
185 190 Lys Lys Glu Ala Asn Ala Thr Glu Asp Asp Leu Leu Gly Arg Gln
Ile 195 200 205 Leu Lys Gln Arg Glu Ser Gly Glu Ala Asp His Gly Glu
Leu Val Gly 210 215 220 Leu Ala Phe Leu Leu Leu Ile Ala Gly His Glu
Thr Thr Ala Asn Met 225 230 235 240 Ile Ser Leu Gly Thr Val Thr Leu
Leu Glu Asn Pro Asp Gln Leu Ala 245 250 255 Lys Ile Lys Ala Asp Pro
Gly Lys Thr Leu Ala Ala Ile Glu Glu Leu 260 265 270 Leu Arg Ile Phe
Thr Ile Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr 275 280 285 Ala Asp
Val Glu Ile Gly Gly Thr Leu Ile Arg Ala Gly Glu Gly Val 290 295 300
Val Gly Leu Ser Asn Ala Gly Asn His Asp Pro Asp Gly Phe Glu Asn 305
310 315 320 Pro Asp Thr Phe Asp Ile Glu Arg Gly Ala Arg His His Val
Ala Phe 325 330 335 Gly Phe Gly Val His Gln Cys Leu Gly Gln Asn Leu
Ala Arg Leu Glu 340 345 350 Leu Gln Ile Val Phe Asp Thr Leu Phe Arg
Arg Val Pro Gly Ile Arg 355 360 365 Ile Ala Val Pro Val Asp Glu Leu
Pro Phe Lys His Asp Ser Thr Ile 370 375 380 Tyr Gly Leu Arg Ala Leu
Pro Val Thr Trp 385 390 48 274 PRT Amycolatopsis orientalis 48 Ser
Gly Gln Thr Ala Trp Ala Leu Thr Arg Leu Glu Asp Ile Arg Glu 1 5 10
15 Met Leu Ser Ser Pro His Phe Ser Ser Asp Arg Gln Asn Pro Ser Phe
20 25 30 Pro Leu Met Val Ala Arg Gln Ile Arg Arg Glu Asp Lys Pro
Phe Arg 35 40 45 Pro Ser Leu Ile Ala Met Asp Pro Pro Glu His Ser
Lys Ala Arg Arg 50 55 60 Asp Val Val Gly Glu Phe Thr Val Lys Arg
Met Lys Ala Leu Gln Pro 65 70 75 80 Arg Ile Gln Gln Ile Val Asp Glu
His Ile Asp Ala Leu Leu Ala Gly 85 90 95 Pro Lys Pro Ala Asp Leu
Val Gln Ala Leu Ser Leu Pro Val Pro Ser 100 105 110 Leu Val Ile Cys
Glu Leu Leu Gly Val Pro Tyr Ser Asp His Glu Phe 115 120 125 Phe Gln
Ser Cys Ser Ser Arg Met Leu Ser Arg Glu Val Thr Ala Glu 130 135 140
Glu Arg Met Thr Ala Phe Glu Ser Leu Glu Asn Tyr Leu Asp Glu Leu 145
150 155 160 Val Thr Lys Lys Glu Ala Asn Ala Thr Glu Asp Asp Leu Leu
Gly Arg 165 170 175 Gln Ile Leu Lys Gln Arg Glu Thr Gly Glu Ala Asp
His Gly Glu Leu 180 185 190 Val Gly Leu Ala Phe Leu Leu Leu Ile Ala
Gly His Glu Thr Thr Ala 195 200 205 Asn Met Ile Ser Leu Gly Thr Ala
Thr Leu Leu Glu Asn Pro Asp Gln 210 215 220 Leu Ala Lys Ile Lys Ala
Asp Pro Gly Lys Thr Leu Ala Ala Ile Glu 225 230 235 240 Glu Leu Leu
Arg Val Phe Thr Ile Ala Glu Thr Ala Thr Ser Arg Phe 245 250 255 Ala
Thr Ala Asp Val Glu Ile Gly Gly Thr Leu Ile Arg Ala Gly Glu 260 265
270 Gly Val 49 367 PRT Amycolatopsis orientalis 49 Ala Thr Leu Pro
Leu Ala Arg Lys Cys Pro Phe Ser Pro Pro Pro Glu 1 5 10 15 Tyr Glu
Arg Leu Arg Arg Glu Ser Pro Val Ser Arg Val Gly Leu Pro 20 25 30
Ser Gly Gln Thr Ala Trp Ala Leu Thr Arg Leu Glu Asp Ile Arg Glu 35
40 45 Met Leu Ser Ser Pro His Phe Ser Ser Asp Arg Gln Ser Pro Ser
Phe 50 55 60 Pro Leu Met Val Ala Arg Gln Ile Arg Arg Glu Asp Lys
Pro Phe Arg 65 70 75 80 Pro Ser Leu Ile Ala Met Asp Pro Pro Glu His
Gly Lys Ala Arg Arg 85 90 95 Asp Val Val Gly Glu Phe Thr Val Lys
Arg Met Lys Ala Leu Gln Pro 100 105 110 Arg Ile Gln Gln Ile Val Asp
Glu His Ile Asp Ala Leu Leu Ala Gly 115 120 125 Pro Lys Pro Ala Asp
Leu Val Gln Ala Leu Ser Leu Pro Val Pro Ser 130 135 140 Leu Val Ile
Cys Glu Leu Leu Gly Val Pro Tyr Ser Asp His Glu Phe 145 150 155 160
Phe Gln Ser Cys Ser Ser Arg Met Leu Ser Arg Glu Val Thr Ala Glu 165
170 175 Glu Arg Met Thr Ala Phe Glu Ser Leu Glu Asn Tyr Leu Asp Glu
Leu 180 185 190 Val Thr Lys Lys Glu Ala Asn Ala Thr Glu Asp Asp Leu
Leu Gly Arg 195 200 205 Gln Ile Leu Lys Gln Arg Glu Ser Gly Glu Ala
Asp His Gly Glu Leu 210 215 220 Val Gly Leu Ala Phe Leu Leu Leu Ile
Ala Gly His Glu Thr Thr Ala 225 230 235 240 Asn Met Ile Ser Leu Gly
Thr Val Thr Leu Leu Glu Asn Pro Asp Gln 245 250 255 Leu Ala Lys Ile
Lys Ala Asp Pro Gly Lys Thr Leu Ala Ala Ile Glu 260 265 270 Glu Leu
Leu Arg Ile Phe Thr Ile Ala Glu Thr Ala Thr Ser Arg Phe 275 280 285
Ala Thr Ala Asp Val Glu Ile Gly Gly Thr Leu Ile Arg Ala Gly Glu 290
295 300 Gly Val Val Gly Leu Ser Asn Ala Gly Asn His Asp Pro Asp Gly
Phe 305 310 315 320 Glu Asn Pro Asp Thr Phe Asp Ile Glu Arg Gly Ala
Arg His His Val 325 330 335 Ala Phe Gly Phe Gly Val His Gln Cys Leu
Gly Gln Asn Leu Ala Arg 340 345 350 Leu Glu Leu Gln Ile Val Phe Asp
Thr Leu Phe Arg Arg Val Pro 355 360 365 50 25 DNA Artificial
sequence Synthetic 50 aggaaaccac cgcgaccttg ccact 25 51 25 DNA
Artificial sequence Synthetic 51 accgaatccg aaggcgacgt gatgc 25 52
23 DNA Artificial sequence Synthetic 52 cggaatgaat ccatccgcat acg
23 53 23 DNA Artificial sequence Synthetic 53 tgatcttcat ggctcctcct
acc 23 54 35 DNA Artificial sequence Synthetic 54 gcgaagccga
ccacggcnnn ctggtcggtc tggcg 35 55 35 DNA Artificial sequence
Synthetic 55 cgccagaccg accagnnngc cgtggtcggc ttcgc 35 56 35 DNA
Artificial sequence Synthetic 56 ggtcggtctg gcgnysctcc tgctcatcgc
ggggc 35 57 35 DNA Artificial sequence Synthetic 57 gccccgcgat
gagcaggags rncgccagac cgacc 35 58 35 DNA Artificial sequence
Synthetic 58 ggtcggtctg gcgttcnysc tgctcatcgc ggggc 35 59 35 DNA
Artificial sequence Synthetic 59 gccccgcgat gagcagsrng aacgccagac
cgacc 35 60 1215 DNA Artificial sequence Synthetic 60 atgaccgacg
tcgaggaaac caccgcgacc ttgccactgg cccgcaaatg cccgttttca 60
ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg tttcccgggt cggtctcccc
120 tccggtcaaa ccgcttgggc gctcacccgg ctcgaagaca tccgcgaaat
gctgagcagt 180 ccgcatttca gctccgacca gcagagtccg tcgttcccgc
tgatggtggc gcggcagatc 240 cggcgcgagg acaagccgtt ccgcccgtcc
ctcgtcgcga tggacccgcc ggaacacggc 300 aaggccaggc gtgacgtcgt
cggggaattc accgtcaagc gcatgaaagc gcttcagcca 360 cgtattcagc
agatcgtcga cgagcatatc gacgccctgc tcgccggccc caaacccgcc 420
gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg tgatctgcga actgctcggt
480 gtcccctatt cggaccacga gttcttccag tcctgcagtt cccggatgct
cagccgggaa 540 gtcaccgccg aagaacggat gaccgcgttc gagtcgctcg
agaactatct cgacgaactc 600 gtcacgaaga aggaggcgaa cgccaccgag
gacgacctcc tcggccgcca gatcctgaag 660 cagcgcgaat ccggcgaagc
cgaccacggc gaactggtcg gtctggcggc gctcctgctc 720 atcgcggggc
acgagactac ggcgaacatg atctcgctcg gcacggtgac cctgctggag 780
aaccccgatc agctggcgaa gatcaaggcg gatccgggca agaccctcgc cgcgatcgag
840 gaactcctgc ggatcttcac catcgcggag acggcgacct cacgcttcgc
cacggcggac 900 gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg
tcgtcggcct gagcaacgcg 960 ggcaaccacg atccggacgg cttcgagaac
ccggacacct tcgacatcga acgcggcgcg 1020 cggcatcacg tcgccttcgg
attcggtgtg caccaatgcc tcggccagaa cttggcgagg 1080 ttggaactcc
agatcgtgtt cgatacgttg ttccggcgag tgccgggcat ccggatcgcc 1140
gtaccggtcg acgaactgcc gttcaagcac gattcgacga tctacggcct ccacgccctg
1200 ccggtcacct ggtag 1215 61 404 PRT Artificial sequence Synthetic
61 Met Thr Asp Val Glu Glu Thr Thr Ala Thr Leu Pro Leu Ala Arg Lys
1 5 10 15 Cys Pro Phe Ser Pro Pro Pro Glu Tyr Glu Arg Leu Arg Arg
Glu Ser 20 25 30 Pro Val Ser Arg Val Gly Leu Pro Ser Gly Gln Thr
Ala Trp Ala Leu 35 40 45 Thr Arg Leu Glu Asp Ile Arg Glu Met Leu
Ser Ser Pro His Phe Ser 50 55 60 Ser Asp Gln Gln Ser Pro Ser Phe
Pro Leu Met Val Ala Arg Gln Ile 65 70 75 80 Arg Arg Glu Asp Lys Pro
Phe Arg Pro Ser Leu Val Ala Met Asp Pro 85 90 95 Pro Glu His Gly
Lys Ala Arg Arg Asp Val Val Gly Glu Phe Thr Val 100 105 110 Lys Arg
Met Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile Val Asp Glu 115 120 125
His Ile Asp Ala Leu Leu Ala Gly Pro Lys Pro Ala Asp Leu Val Gln 130
135 140 Ala Leu Ser Leu Pro Val Pro Ser Leu Val Ile Cys Glu Leu Leu
Gly 145 150 155 160 Val Pro Tyr Ser Asp His Glu Phe Phe Gln Ser Cys
Ser Ser Arg Met 165 170 175 Leu Ser Arg Glu Val Thr Ala Glu Glu Arg
Met Thr Ala Phe Glu Ser 180 185 190 Leu Glu Asn Tyr Leu Asp Glu Leu
Val Thr Lys Lys Glu Ala Asn Ala 195 200 205 Thr Glu Asp Asp Leu Leu
Gly Arg Gln Ile Leu Lys Gln Arg Glu Ser 210 215 220 Gly Glu Ala Asp
His Gly Glu Leu Val Gly Leu Ala Ala Leu Leu Leu 225 230 235 240 Ile
Ala Gly His Glu Thr Thr Ala Asn Met Ile Ser Leu Gly Thr Val 245
250
255 Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala Lys Ile Lys Ala Asp Pro
260 265 270 Gly Lys Thr Leu Ala Ala Ile Glu Glu Leu Leu Arg Ile Phe
Thr Ile 275 280 285 Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr Ala Asp
Val Glu Ile Gly 290 295 300 Gly Thr Leu Ile Arg Ala Gly Glu Gly Val
Val Gly Leu Ser Asn Ala 305 310 315 320 Gly Asn His Asp Pro Asp Gly
Phe Glu Asn Pro Asp Thr Phe Asp Ile 325 330 335 Glu Arg Gly Ala Arg
His His Val Ala Phe Gly Phe Gly Val His Gln 340 345 350 Cys Leu Gly
Gln Asn Leu Ala Arg Leu Glu Leu Gln Ile Val Phe Asp 355 360 365 Thr
Leu Phe Arg Arg Val Pro Gly Ile Arg Ile Ala Val Pro Val Asp 370 375
380 Glu Leu Pro Phe Lys His Asp Ser Thr Ile Tyr Gly Leu His Ala Leu
385 390 395 400 Pro Val Thr Trp 62 1215 DNA Artificial sequence
Synthetic 62 atgaccgacg tcgaggaaac caccgcgacc ttgccactgg cccgcaaatg
cccgttttca 60 ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg
tttcccgggt cggtctcccc 120 tccggtcaaa ccgcttgggc gctcacccgg
ctcgaagaca tccgcgaaat gctgagcagt 180 ccgcatttca gctccgaccg
gcagagtccg tcgttcccgc tgatggtggc gcggcagatc 240 cggcgcgagg
acaagccgtt ccgcccgtcc ctcgtcggga tggacccgcc ggaacacggc 300
aaggccaggc gtgacgtcgt cggggaattc accgtcaagc gcatgaaagc gcttcagcca
360 cgtattcagc agatcgtcga cgagcatatc gacgccctgc tcgccggccc
caaacccgcc 420 gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg
tgatctgcga actgctcggt 480 gtcccctatt cggaccacga gttcttccag
tcctgcagtt cccggatgct cagccgggaa 540 gtcaccgccg aagaacggat
gaccgcgttc gagtcgctcg agaactatct cgacgaactc 600 gtcacgaaga
aggaggcgaa cgccaccgag gacgacctcc tcggccgcca gatcctgaag 660
cagcgcgaat ccggcgaagc cgaccacggc gaactggtcg gtctggcggc gctcctgctc
720 atcgcggggc acgagactac ggcgaacatg atctcgctcg gcacggtgac
cctgctggag 780 aaccccgatc agctggcgaa gatcaaggcg gatccgggca
agaccctcgc cgcgatcgag 840 gaactcctgc ggatcttcac catcgcggag
acggcgacct cacgcttcgc cacggcggac 900 gtcgagatcg gcggcacgct
catccgcgcg ggtgaaggcg tcgtcggcct gagcaacgcg 960 ggcaaccacg
atccggacgg cttcgagaac ccggacacct tcgacatcga acgcggcgcg 1020
cggcatcacg tcgccttcgg attcggtgtg caccaatgcc tcggccagaa cttggcgagg
1080 ttggaactcc agaccgtgtt cgatacgttg ttccggcgag tgccgggcat
ccggatcgcc 1140 gtaccggtcg acgaactgcc gttcaagcac gattcgacga
tctacggcct ccacgccctg 1200 ccggtcacct ggtag 1215 63 404 PRT
Artificial sequence Synthetic 63 Met Thr Asp Val Glu Glu Thr Thr
Ala Thr Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro Phe Ser Pro Pro
Pro Glu Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30 Pro Val Ser Arg
Val Gly Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35 40 45 Thr Arg
Leu Glu Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe Ser 50 55 60
Ser Asp Arg Gln Ser Pro Ser Phe Pro Leu Met Val Ala Arg Gln Ile 65
70 75 80 Arg Arg Glu Asp Lys Pro Phe Arg Pro Ser Leu Val Gly Met
Asp Pro 85 90 95 Pro Glu His Gly Lys Ala Arg Arg Asp Val Val Gly
Glu Phe Thr Val 100 105 110 Lys Arg Met Lys Ala Leu Gln Pro Arg Ile
Gln Gln Ile Val Asp Glu 115 120 125 His Ile Asp Ala Leu Leu Ala Gly
Pro Lys Pro Ala Asp Leu Val Gln 130 135 140 Ala Leu Ser Leu Pro Val
Pro Ser Leu Val Ile Cys Glu Leu Leu Gly 145 150 155 160 Val Pro Tyr
Ser Asp His Glu Phe Phe Gln Ser Cys Ser Ser Arg Met 165 170 175 Leu
Ser Arg Glu Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu Ser 180 185
190 Leu Glu Asn Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu Ala Asn Ala
195 200 205 Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu Lys Gln Arg
Glu Ser 210 215 220 Gly Glu Ala Asp His Gly Glu Leu Val Gly Leu Ala
Ala Leu Leu Leu 225 230 235 240 Ile Ala Gly His Glu Thr Thr Ala Asn
Met Ile Ser Leu Gly Thr Val 245 250 255 Thr Leu Leu Glu Asn Pro Asp
Gln Leu Ala Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys Thr Leu Ala
Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr Ile 275 280 285 Ala Glu Thr
Ala Thr Ser Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290 295 300 Gly
Thr Leu Ile Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn Ala 305 310
315 320 Gly Asn His Asp Pro Asp Gly Phe Glu Asn Pro Asp Thr Phe Asp
Ile 325 330 335 Glu Arg Gly Ala Arg His His Val Ala Phe Gly Phe Gly
Val His Gln 340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg Leu Glu Leu
Gln Thr Val Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val Pro Gly Ile
Arg Ile Ala Val Pro Val Asp 370 375 380 Glu Leu Pro Phe Lys His Asp
Ser Thr Ile Tyr Gly Leu His Ala Leu 385 390 395 400 Pro Val Thr Trp
64 1215 DNA Artificial sequence Synthetic 64 atgaccgacg tcgaggaaac
caccgcgacc ttgccactgg cccgcaaatg cccgttttca 60 ccaccgcccg
aatacgagcg gctccgccgg gaaagtccgg tttcccgggt cggtctcccc 120
tccggtcaaa ccgcttgggc gctcacccgg ctcgaagaca tccgcgaaat gctgagcagt
180 ccgcatttca gctccgaccg gcagagtccg tcgttcccgc tgatggtggc
gcggcagatc 240 cggcgcgagg acaagccgtt ccgcccgtcc ctcgtcgcga
tggacccgcc ggaacacggc 300 aaggccaggc gtgacgccgt cggggaattc
accgtcaagc gcatgaaagc gcttcagcca 360 cgtattcagc agatcgtcga
cgagcatatc gacgccctgc tcgccggccc caaacccgcc 420 gatctcgtcc
aggcgctttc cctgccggtt ccgtccttgg tgatctgcga actgctcggt 480
gtcccctatt cggaccacga gttcttccag tcctgcagtt cccggatgct cagccgggaa
540 gtcaccgccg aagaacggat gaccgcgttc gagtcgctcg agaactatct
cgacgaactc 600 gtcacgaaga aggaggcgaa cgccaccgag gacgacctcc
tcggccgcca gatcctgaag 660 cagcgcgaat ccggcgaagc cgaccacggc
gaactggtcg gtctggcggc gctcctgctc 720 atcgcggggc acgagactac
ggcgaacatg atctcgctcg gcacggtgac cctgctggag 780 aaccccgatc
agctggcgaa gatcaaggca gatccgggca agaccctcgc cgcgatcgag 840
gaactcctgc ggatcttcac catcgcggag acggcgacct cacgcttcgc cacggcggac
900 gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg tcgtcggcct
gagcaacgcg 960 ggcaaccacg atccggacgg cttcgagaac ccggacacct
tcgacatcga acgcggcgcg 1020 cggcatcacg tcgccttcgg attcggtgtg
caccaatgcc tcggccagaa cttggcgagg 1080 ttggaactcc agatcgtgtt
cgatacgttg ttccggcgag tgccgggcat ccggatcgcc 1140 gtaccggtcg
acgaactgcc gttcaagcac gattcgacga tctacggcct ccacgccctg 1200
ccggtcacct ggtag 1215 65 404 PRT Artificial sequence Synthetic 65
Met Thr Asp Val Glu Glu Thr Thr Ala Thr Leu Pro Leu Ala Arg Lys 1 5
10 15 Cys Pro Phe Ser Pro Pro Pro Glu Tyr Glu Arg Leu Arg Arg Glu
Ser 20 25 30 Pro Val Ser Arg Val Gly Leu Pro Ser Gly Gln Thr Ala
Trp Ala Leu 35 40 45 Thr Arg Leu Glu Asp Ile Arg Glu Met Leu Ser
Ser Pro His Phe Ser 50 55 60 Ser Asp Arg Gln Ser Pro Ser Phe Pro
Leu Met Val Ala Arg Gln Ile 65 70 75 80 Arg Arg Glu Asp Lys Pro Phe
Arg Pro Ser Leu Val Ala Met Asp Pro 85 90 95 Pro Glu His Gly Lys
Ala Arg Arg Asp Ala Val Gly Glu Phe Thr Val 100 105 110 Lys Arg Met
Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile Val Asp Glu 115 120 125 His
Ile Asp Ala Leu Leu Ala Gly Pro Lys Pro Ala Asp Leu Val Gln 130 135
140 Ala Leu Ser Leu Pro Val Pro Ser Leu Val Ile Cys Glu Leu Leu Gly
145 150 155 160 Val Pro Tyr Ser Asp His Glu Phe Phe Gln Ser Cys Ser
Ser Arg Met 165 170 175 Leu Ser Arg Glu Val Thr Ala Glu Glu Arg Met
Thr Ala Phe Glu Ser 180 185 190 Leu Glu Asn Tyr Leu Asp Glu Leu Val
Thr Lys Lys Glu Ala Asn Ala 195 200 205 Thr Glu Asp Asp Leu Leu Gly
Arg Gln Ile Leu Lys Gln Arg Glu Ser 210 215 220 Gly Glu Ala Asp His
Gly Glu Leu Val Gly Leu Ala Ala Leu Leu Leu 225 230 235 240 Ile Ala
Gly His Glu Thr Thr Ala Asn Met Ile Ser Leu Gly Thr Val 245 250 255
Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala Lys Ile Lys Ala Asp Pro 260
265 270 Gly Lys Thr Leu Ala Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr
Ile 275 280 285 Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr Ala Asp Val
Glu Ile Gly 290 295 300 Gly Thr Leu Ile Arg Ala Gly Glu Gly Val Val
Gly Leu Ser Asn Ala 305 310 315 320 Gly Asn His Asp Pro Asp Gly Phe
Glu Asn Pro Asp Thr Phe Asp Ile 325 330 335 Glu Arg Gly Ala Arg His
His Val Ala Phe Gly Phe Gly Val His Gln 340 345 350 Cys Leu Gly Gln
Asn Leu Ala Arg Leu Glu Leu Gln Ile Val Phe Asp 355 360 365 Thr Leu
Phe Arg Arg Val Pro Gly Ile Arg Ile Ala Val Pro Val Asp 370 375 380
Glu Leu Pro Phe Lys His Asp Ser Thr Ile Tyr Gly Leu His Ala Leu 385
390 395 400 Pro Val Thr Trp 66 1215 DNA Artificial sequence
Synthetic 66 atgaccgacg tcgaggaaac caccgcgacc ttgccactgg ctcgcaaatg
cccgttttca 60 ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg
tttcccgggt cggtctcccc 120 tccggtcaaa ccgcttgggc gctcacccgg
ctcgaagaca tccgcgaaat gctgagcagt 180 ccgcatttca gctccgaccg
gcagagtccg tcgttcccgc tgatggtggc gcggcagatc 240 cggcgcgagg
acaagccgtt ccacccgtcc ctcgtcgcga tggacccgcc ggaacacggc 300
aaggccaggc gtgacgtcgt cggggaattc accgtcaagc gcatgaaagc gcttcagcca
360 cgtattcagc agatcgtcga cgagcatatc gacgccctgc tcgccggccc
caaacccgcc 420 gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg
tgatctgcga actgctcggt 480 gtcccctatt cggaccacga gttcttccag
tcctgcagtt cccggatgct cagccgggaa 540 gtcaccgccg aagaacggat
gaccgcgttc gagtcgctcg agaactatct cgacgaactc 600 gtcacgaaga
aggaggcgaa cgccaccgag gacgacctcc tcggccgcca gatcctgaag 660
cagcgcgaat ccggcgaagc cgaccacggc gaactggtcg gtctggcggc gctcctgctc
720 atcgcggggc acgagactac ggcgaacatg atctcgctcg gcacggtgac
cctgctggag 780 aaccccgatc agctggcgaa gatcaaggcg gatccgggca
agaccctcgc cgcgatcgag 840 gaactcctgc ggatcttcac catcgcggag
acggcgacct cacgcttcgc cacggcggac 900 gtcgagatcg gcggcacgct
catccgcgcg ggtgaaggcg tcgtcggcct gagcaacgcg 960 ggcaaccacg
atccggacgg cttcgagaac ccggacacct tcgacatcga acgcggcgcg 1020
cggcatcacg tcgccttcgg attcggtgtg caccaatgcc tcggccagaa cttggcgagg
1080 ttggaactcc agatcgtgtt cgatacgttg ttccggcgag tgccgggcat
ccggatcgcc 1140 gtaccggtcg acgaactgcc gttcaagcac gattcgacga
tctacggcct ccacgccctg 1200 ccggtcacct ggtag 1215 67 404 PRT
Artificial sequence Synthetic 67 Met Thr Asp Val Glu Glu Thr Thr
Ala Thr Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro Phe Ser Pro Pro
Pro Glu Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30 Pro Val Ser Arg
Val Gly Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35 40 45 Thr Arg
Leu Glu Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe Ser 50 55 60
Ser Asp Arg Gln Ser Pro Ser Phe Pro Leu Met Val Ala Arg Gln Ile 65
70 75 80 Arg Arg Glu Asp Lys Pro Phe His Pro Ser Leu Val Ala Met
Asp Pro 85 90 95 Pro Glu His Gly Lys Ala Arg Arg Asp Val Val Gly
Glu Phe Thr Val 100 105 110 Lys Arg Met Lys Ala Leu Gln Pro Arg Ile
Gln Gln Ile Val Asp Glu 115 120 125 His Ile Asp Ala Leu Leu Ala Gly
Pro Lys Pro Ala Asp Leu Val Gln 130 135 140 Ala Leu Ser Leu Pro Val
Pro Ser Leu Val Ile Cys Glu Leu Leu Gly 145 150 155 160 Val Pro Tyr
Ser Asp His Glu Phe Phe Gln Ser Cys Ser Ser Arg Met 165 170 175 Leu
Ser Arg Glu Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu Ser 180 185
190 Leu Glu Asn Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu Ala Asn Ala
195 200 205 Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu Lys Gln Arg
Glu Ser 210 215 220 Gly Glu Ala Asp His Gly Glu Leu Val Gly Leu Ala
Ala Leu Leu Leu 225 230 235 240 Ile Ala Gly His Glu Thr Thr Ala Asn
Met Ile Ser Leu Gly Thr Val 245 250 255 Thr Leu Leu Glu Asn Pro Asp
Gln Leu Ala Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys Thr Leu Ala
Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr Ile 275 280 285 Ala Glu Thr
Ala Thr Ser Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290 295 300 Gly
Thr Leu Ile Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn Ala 305 310
315 320 Gly Asn His Asp Pro Asp Gly Phe Glu Asn Pro Asp Thr Phe Asp
Ile 325 330 335 Glu Arg Gly Ala Arg His His Val Ala Phe Gly Phe Gly
Val His Gln 340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg Leu Glu Leu
Gln Ile Val Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val Pro Gly Ile
Arg Ile Ala Val Pro Val Asp 370 375 380 Glu Leu Pro Phe Lys His Asp
Ser Thr Ile Tyr Gly Leu His Ala Leu 385 390 395 400 Pro Val Thr Trp
68 1215 DNA Artificial sequence Synthetic 68 atgaccgacg tcgaggaaac
caccgcgacc ttgccactgg cccgcaaatg cccgttttca 60 ccaccgcccg
aatacgagcg gctccgccgg aaaagtccgg tttcccgggt cggtctcccc 120
tccggtcaaa ccgcttgggc gctcacccgg ctcgaagaca tccgcgaaat gctgagcagt
180 ccgcatttca gctccgaccg gcagagtccg tcgttcccgc tgatggtggc
gcggcagatc 240 cggcgcgagg acaagccgtt ccgcccgtcc ctcatcgcga
tggacccgcc ggaacacggc 300 aaggccaggc gtgacgtcgt cggggaattc
accgtcaagc gcatgaaagc gcttcagcca 360 cgtattcagc agatcgtcga
cgagcatatc gacgccctgc tcgccggccc caaacccgcc 420 gatctcgtcc
aggcgctttc cctgccggtt ccgtccttgg tgatctgcga actgctcggt 480
gtcccctatt cggaccacga gttcttccag tcctgcagtt cccggatgct cagccgggaa
540 gtcaccgccg aagaacggat gaccgcgttc gagtcgctcg agaactatct
cgacgaactc 600 gtcacgaaga aggaggcgaa cgccaccgag gacgacctcc
tcggccgcca gatcctgaag 660 cagcgcgaat ccggcgaagc cgaccacggc
gaactggtcg gtctggcgtt cctcctgctc 720 atcgcggggc acgagactac
ggcgaacatg atctcgctcg gcacggtgac cctgctggag 780 aaccccgatc
agctggcgaa gatcaaggcg gatccgggca agaccctcgc cgcgatcgag 840
gaactcctgc ggatcttcac catcgcggag acggcgacct cacgcttcgc cacggcggac
900 gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg tcgtcggcct
gagcaacgcg 960 ggcaaccacg atccggacgg cttcgagaac ccggacacct
tcgacatcga acgcggcgcg 1020 cggcatcacg tcgccttcgg attcggtgtg
caccaatgcc tcggccagaa cttggcgagg 1080 ttggaactcc agatcgtgtt
cgatacgttg ttccggcgag tgccgggcat ccggatcgcc 1140 gtaccggtcg
acgaactgcc gttcaagcac gattcgacga tctacggcct ccacgccctg 1200
ccggtcacct ggtag 1215 69 404 PRT Artificial sequence Synthetic 69
Met Thr Asp Val Glu Glu Thr Thr Ala Thr Leu Pro Leu Ala Arg Lys 1 5
10 15 Cys Pro Phe Ser Pro Pro Pro Glu Tyr Glu Arg Leu Arg Arg Lys
Ser 20 25 30 Pro Val Ser Arg Val Gly Leu Pro Ser Gly Gln Thr Ala
Trp Ala Leu 35 40 45 Thr Arg Leu Glu Asp Ile Arg Glu Met Leu Ser
Ser Pro His Phe Ser 50 55 60 Ser Asp Arg Gln Ser Pro Ser Phe Pro
Leu Met Val Ala Arg Gln Ile 65 70 75 80 Arg Arg Glu Asp Lys Pro Phe
Arg Pro Ser Leu Ile Ala Met Asp Pro 85 90 95 Pro Glu His Gly Lys
Ala Arg Arg Asp Val Val Gly Glu Phe Thr Val 100 105 110 Lys Arg Met
Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile Val Asp Glu 115 120 125 His
Ile Asp Ala Leu Leu Ala Gly Pro Lys Pro Ala Asp Leu Val Gln 130 135
140 Ala Leu Ser Leu Pro Val Pro Ser Leu Val Ile Cys Glu Leu Leu Gly
145 150 155 160 Val Pro Tyr Ser Asp His Glu Phe Phe Gln Ser Cys Ser
Ser Arg Met 165 170 175 Leu Ser Arg Glu Val Thr Ala Glu Glu Arg Met
Thr Ala Phe Glu Ser 180 185 190 Leu Glu Asn Tyr Leu Asp Glu Leu Val
Thr Lys Lys Glu Ala Asn Ala 195 200 205 Thr Glu Asp Asp Leu Leu Gly
Arg Gln Ile Leu Lys Gln Arg Glu Ser 210 215 220 Gly Glu Ala Asp
His Gly Glu Leu Val Gly Leu Ala Phe Leu Leu Leu 225 230 235 240 Ile
Ala Gly His Glu Thr Thr Ala Asn Met Ile Ser Leu Gly Thr Val 245 250
255 Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala Lys Ile Lys Ala Asp Pro
260 265 270 Gly Lys Thr Leu Ala Ala Ile Glu Glu Leu Leu Arg Ile Phe
Thr Ile 275 280 285 Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr Ala Asp
Val Glu Ile Gly 290 295 300 Gly Thr Leu Ile Arg Ala Gly Glu Gly Val
Val Gly Leu Ser Asn Ala 305 310 315 320 Gly Asn His Asp Pro Asp Gly
Phe Glu Asn Pro Asp Thr Phe Asp Ile 325 330 335 Glu Arg Gly Ala Arg
His His Val Ala Phe Gly Phe Gly Val His Gln 340 345 350 Cys Leu Gly
Gln Asn Leu Ala Arg Leu Glu Leu Gln Ile Val Phe Asp 355 360 365 Thr
Leu Phe Arg Arg Val Pro Gly Ile Arg Ile Ala Val Pro Val Asp 370 375
380 Glu Leu Pro Phe Lys His Asp Ser Thr Ile Tyr Gly Leu His Ala Leu
385 390 395 400 Pro Val Thr Trp 70 35 DNA Artificial sequence
Synthetic 70 gttccgcccg tccctcgtcn nsatggaccc gccgg 35 71 35 DNA
Artificial sequence Synthetic 71 cctgcagttc ccggnnsctc agccgggaag
tcacc 35 72 1215 DNA Artificial sequence Synthetic 72 atgaccgacg
tcgaggaaac caccgcgacc ttgccactgg cccgcaaatg cccgttttca 60
ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg tttcccgggt cggtctcccc
120 tccggtcaga ccgcttgggc gctcacccgg ctcgaagaca tccgcgaaat
gctgagcagt 180 ccgcatttca gctccgacca gcagagtccg tcgttcccgc
tgatggtggc gcggcagatc 240 cggcgcgagg acaagccgtt ccgcccgtcc
ctcgtcgcga tggacccgcc ggaacacggc 300 aaggccaggc gtgacgtcgt
cggggaattc accgtcaagc gcatgaaagc gcttcagcca 360 cgtattcagc
agatcgtcga cgagcatacc gacgccctgc tcgccggccc caaacccgcc 420
gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg tgatctgcga actgctcggt
480 gtcccctatt cggaccacga gttcttccag tcctgcagtt cccgggcgct
cagccgggaa 540 gtcaccgccg aagaacggat gaccgcgttc gagtcgctcg
agaactatct cgacgaactc 600 gtcacgaaga aggaggcgaa cgccaccgag
gacgacctcc tcggccgcca gatcctgaag 660 cagcgcgaat ccggcgaagc
cgaccacggc gaactggtcg gtctggcggc gctcctgctc 720 atcgcggggc
acgagactac ggcgaacatg atctcgctcg gcacggtgac cctgctggag 780
aaccccgatc agctggcgaa gatcaaggcg gacccgggca agaccctcgc cgcgatcgag
840 gaactcctgc ggatcttcac catcgcggag acggcgacct cacgcttcgc
cacggcggac 900 gtcgagatcg gcggcacgct catccgcgcg ggtgaaggcg
tcgtcggcct gagcaacgcg 960 ggcaaccacg atccggacgg cttcgagaac
ccggacacct tcgacatcga acgcggcgcg 1020 cggcatcacg tcgccttcgg
attcggtgtg caccaatgcc tcggccagaa cttggcgagg 1080 ttggaactcc
agatcgtgtt cgatacgttg ttccggcgag tgccgggcat ccggatcgcc 1140
gtaccggtcg acgaactgcc gttcaagcac gattcgacga tctacggcct ccacgccctg
1200 ccggtcacct ggtag 1215 73 404 PRT Artificial sequence Synthetic
73 Met Thr Asp Val Glu Glu Thr Thr Ala Thr Leu Pro Leu Ala Arg Lys
1 5 10 15 Cys Pro Phe Ser Pro Pro Pro Glu Tyr Glu Arg Leu Arg Arg
Glu Ser 20 25 30 Pro Val Ser Arg Val Gly Leu Pro Ser Gly Gln Thr
Ala Trp Ala Leu 35 40 45 Thr Arg Leu Glu Asp Ile Arg Glu Met Leu
Ser Ser Pro His Phe Ser 50 55 60 Ser Asp Gln Gln Ser Pro Ser Phe
Pro Leu Met Val Ala Arg Gln Ile 65 70 75 80 Arg Arg Glu Asp Lys Pro
Phe Arg Pro Ser Leu Val Ala Met Asp Pro 85 90 95 Pro Glu His Gly
Lys Ala Arg Arg Asp Val Val Gly Glu Phe Thr Val 100 105 110 Lys Arg
Met Lys Ala Leu Gln Pro Arg Ile Gln Gln Ile Val Asp Glu 115 120 125
His Thr Asp Ala Leu Leu Ala Gly Pro Lys Pro Ala Asp Leu Val Gln 130
135 140 Ala Leu Ser Leu Pro Val Pro Ser Leu Val Ile Cys Glu Leu Leu
Gly 145 150 155 160 Val Pro Tyr Ser Asp His Glu Phe Phe Gln Ser Cys
Ser Ser Arg Ala 165 170 175 Leu Ser Arg Glu Val Thr Ala Glu Glu Arg
Met Thr Ala Phe Glu Ser 180 185 190 Leu Glu Asn Tyr Leu Asp Glu Leu
Val Thr Lys Lys Glu Ala Asn Ala 195 200 205 Thr Glu Asp Asp Leu Leu
Gly Arg Gln Ile Leu Lys Gln Arg Glu Ser 210 215 220 Gly Glu Ala Asp
His Gly Glu Leu Val Gly Leu Ala Ala Leu Leu Leu 225 230 235 240 Ile
Ala Gly His Glu Thr Thr Ala Asn Met Ile Ser Leu Gly Thr Val 245 250
255 Thr Leu Leu Glu Asn Pro Asp Gln Leu Ala Lys Ile Lys Ala Asp Pro
260 265 270 Gly Lys Thr Leu Ala Ala Ile Glu Glu Leu Leu Arg Ile Phe
Thr Ile 275 280 285 Ala Glu Thr Ala Thr Ser Arg Phe Ala Thr Ala Asp
Val Glu Ile Gly 290 295 300 Gly Thr Leu Ile Arg Ala Gly Glu Gly Val
Val Gly Leu Ser Asn Ala 305 310 315 320 Gly Asn His Asp Pro Asp Gly
Phe Glu Asn Pro Asp Thr Phe Asp Ile 325 330 335 Glu Arg Gly Ala Arg
His His Val Ala Phe Gly Phe Gly Val His Gln 340 345 350 Cys Leu Gly
Gln Asn Leu Ala Arg Leu Glu Leu Gln Ile Val Phe Asp 355 360 365 Thr
Leu Phe Arg Arg Val Pro Gly Ile Arg Ile Ala Val Pro Val Asp 370 375
380 Glu Leu Pro Phe Lys His Asp Ser Thr Ile Tyr Gly Leu His Ala Leu
385 390 395 400 Pro Val Thr Trp 74 1215 DNA Artificial sequence
Synthetic 74 atgaccgacg tcgaggaaac caccgcgacc ttgccactgg cccgcaaatg
cccgttttca 60 ccaccgcccg aatacgagcg gctccgccgg gaaagtccgg
tttcccgggt cggtctcccc 120 tccggtcaaa ccgcttgggc gctcacccgg
ctcgaagaca tccgcgaaat gctgagcagt 180 ccgcatttca gctccgacca
gcagagtccg tcgttcccgc tgatggtggc gcggcagatc 240 cggcgcgagg
acaagccgtt ccgcccgtcc ctcgtcgcga tggacccgcc ggaacacggc 300
aaggccaggc gtgacgtcgt cggggaattc accgtcaagc gcatgaaggc gcttcagcca
360 cgtattcagc agatcgtcga cgagcatatc gacgccctgc tcgccggccc
caaacccacc 420 gatctcgtcc aggcgctttc cctgccggtt ccgtccttgg
tgatctgcga actgctcggt 480 gtcccctatt cggaccacga gttcttccag
tcctgcagtt cccggtcgct cagccgggaa 540 gtcaccgccg aagaacggat
gaccgcgttc gagtcgctcg agaactatct cgacgaactc 600 gtcacgaaga
aggaggcgaa cgccaccgag gacgacctcc tcggccgcca gatcctgaag 660
cagcgcgaat ccggcgaagc cgaccacggc gaactggtcg gtctggcggc gctcctgctc
720 atcgcggggc acgagactac ggcgaacatg atctcgctcg gcacggtgac
cctgctggag 780 aaccccgatc agctggcgaa gatcaaggcg gacccgggca
agaccctcgc cgcgatcgag 840 gaactcctgc ggatcttcac catcgcggag
acggcgacct cacgcttcgc cacggcggac 900 gtcgagatcg gcggcacgct
catccgcgcg ggtgaaggcg tcgtcggcct gagcaacgcg 960 ggcaaccacg
atccggacgg cttcgagaac ccggacacct tcgacatcga acgcggcgcg 1020
cggcatcacg tcgccttcgg attcggtgtg caccaatgcc tcggccagaa cttggcgagg
1080 ttggaactcc agatcgtgtt cgatacgttg ttccggcgag tgccgggcat
ccggatcgcc 1140 gtaccggtcg acgaactgcc gttcaagcac gattcgacga
tctacggcct ccacgccctg 1200 ccggtcacct ggtag 1215 75 404 PRT
Artificial sequence Synthetic 75 Met Thr Asp Val Glu Glu Thr Thr
Ala Thr Leu Pro Leu Ala Arg Lys 1 5 10 15 Cys Pro Phe Ser Pro Pro
Pro Glu Tyr Glu Arg Leu Arg Arg Glu Ser 20 25 30 Pro Val Ser Arg
Val Gly Leu Pro Ser Gly Gln Thr Ala Trp Ala Leu 35 40 45 Thr Arg
Leu Glu Asp Ile Arg Glu Met Leu Ser Ser Pro His Phe Ser 50 55 60
Ser Asp Gln Gln Ser Pro Ser Phe Pro Leu Met Val Ala Arg Gln Ile 65
70 75 80 Arg Arg Glu Asp Lys Pro Phe Arg Pro Ser Leu Val Ala Met
Asp Pro 85 90 95 Pro Glu His Gly Lys Ala Arg Arg Asp Val Val Gly
Glu Phe Thr Val 100 105 110 Lys Arg Met Lys Ala Leu Gln Pro Arg Ile
Gln Gln Ile Val Asp Glu 115 120 125 His Ile Asp Ala Leu Leu Ala Gly
Pro Lys Pro Thr Asp Leu Val Gln 130 135 140 Ala Leu Ser Leu Pro Val
Pro Ser Leu Val Ile Cys Glu Leu Leu Gly 145 150 155 160 Val Pro Tyr
Ser Asp His Glu Phe Phe Gln Ser Cys Ser Ser Arg Ser 165 170 175 Leu
Ser Arg Glu Val Thr Ala Glu Glu Arg Met Thr Ala Phe Glu Ser 180 185
190 Leu Glu Asn Tyr Leu Asp Glu Leu Val Thr Lys Lys Glu Ala Asn Ala
195 200 205 Thr Glu Asp Asp Leu Leu Gly Arg Gln Ile Leu Lys Gln Arg
Glu Ser 210 215 220 Gly Glu Ala Asp His Gly Glu Leu Val Gly Leu Ala
Ala Leu Leu Leu 225 230 235 240 Ile Ala Gly His Glu Thr Thr Ala Asn
Met Ile Ser Leu Gly Thr Val 245 250 255 Thr Leu Leu Glu Asn Pro Asp
Gln Leu Ala Lys Ile Lys Ala Asp Pro 260 265 270 Gly Lys Thr Leu Ala
Ala Ile Glu Glu Leu Leu Arg Ile Phe Thr Ile 275 280 285 Ala Glu Thr
Ala Thr Ser Arg Phe Ala Thr Ala Asp Val Glu Ile Gly 290 295 300 Gly
Thr Leu Ile Arg Ala Gly Glu Gly Val Val Gly Leu Ser Asn Ala 305 310
315 320 Gly Asn His Asp Pro Asp Gly Phe Glu Asn Pro Asp Thr Phe Asp
Ile 325 330 335 Glu Arg Gly Ala Arg His His Val Ala Phe Gly Phe Gly
Val His Gln 340 345 350 Cys Leu Gly Gln Asn Leu Ala Arg Leu Glu Leu
Gln Ile Val Phe Asp 355 360 365 Thr Leu Phe Arg Arg Val Pro Gly Ile
Arg Ile Ala Val Pro Val Asp 370 375 380 Glu Leu Pro Phe Lys His Asp
Ser Thr Ile Tyr Gly Leu His Ala Leu 385 390 395 400 Pro Val Thr Trp
76 404 PRT Saccharopolyspora erythaea 76 Met Thr Thr Val Pro Asp
Leu Glu Ser Asp Ser Phe His Val Asp Trp 1 5 10 15 Tyr Arg Thr Tyr
Ala Glu Leu Arg Glu Thr Ala Pro Val Thr Pro Val 20 25 30 Arg Phe
Leu Gly Gln Asp Ala Trp Leu Val Thr Gly Tyr Asp Glu Ala 35 40 45
Lys Ala Ala Leu Ser Asp Leu Arg Leu Ser Ser Asp Pro Lys Lys Lys 50
55 60 Tyr Pro Gly Val Glu Val Glu Phe Pro Ala Tyr Leu Gly Phe Pro
Glu 65 70 75 80 Asp Val Arg Asn Tyr Phe Ala Thr Asn Met Gly Thr Ser
Asp Pro Pro 85 90 95 Thr His Thr Arg Leu Arg Lys Leu Val Ser Gln
Glu Phe Thr Val Arg 100 105 110 Arg Val Glu Ala Met Arg Pro Arg Val
Glu Gln Ile Thr Ala Glu Leu 115 120 125 Leu Asp Glu Val Gly Asp Ser
Gly Val Val Asp Ile Val Asp Arg Phe 130 135 140 Ala His Pro Leu Pro
Ile Lys Val Ile Cys Glu Leu Leu Gly Val Asp 145 150 155 160 Glu Lys
Tyr Arg Gly Glu Phe Gly Arg Trp Ser Ser Glu Ile Leu Val 165 170 175
Met Asp Pro Glu Arg Ala Glu Gln Arg Gly Gln Ala Ala Arg Glu Val 180
185 190 Val Asn Phe Ile Leu Asp Leu Val Glu Arg Arg Arg Thr Glu Pro
Gly 195 200 205 Asp Asp Leu Leu Ser Ala Leu Ile Arg Val Gln Asp Asp
Asp Asp Gly 210 215 220 Arg Leu Ser Ala Asp Glu Leu Thr Ser Ile Ala
Leu Val Leu Leu Leu 225 230 235 240 Ala Gly Phe Glu Ala Ser Val Ser
Leu Ile Gly Ile Gly Thr Tyr Leu 245 250 255 Leu Leu Thr His Pro Asp
Gln Leu Ala Leu Val Arg Arg Asp Pro Ser 260 265 270 Ala Leu Pro Asn
Ala Val Glu Glu Ile Leu Arg Tyr Ile Ala Pro Pro 275 280 285 Glu Thr
Thr Thr Arg Phe Ala Ala Glu Glu Val Glu Ile Gly Gly Val 290 295 300
Ala Ile Pro Gln Tyr Ser Thr Val Leu Val Ala Asn Gly Ala Ala Asn 305
310 315 320 Arg Asp Pro Lys Gln Phe Pro Asp Pro His Arg Phe Asp Val
Thr Arg 325 330 335 Asp Thr Arg Gly His Leu Ser Phe Gly Gln Gly Ile
His Phe Cys Met 340 345 350 Gly Arg Pro Leu Ala Lys Leu Glu Gly Glu
Val Ala Leu Arg Ala Leu 355 360 365 Phe Gly Arg Phe Pro Ala Leu Ser
Leu Gly Ile Asp Ala Asp Asp Val 370 375 380 Val Trp Arg Arg Ser Leu
Leu Leu Arg Gly Ile Asp His Leu Pro Val 385 390 395 400 Arg Leu Asp
Gly
* * * * *